Methods and compositions for enhanced drug delivery to the eye and extended delivery formulations

ABSTRACT

The present invention comprises compounds and compositions thereof for enhanced drug delivery. Pro-drug and double pro-drug derivatives of corticosteroids non-steroid anti-inflammatory drugs (NSAIDs), and ruboxistaurin for delivery to the eye are provided. The compounds and compositions are useful for treating various ocular diseases, including ocular diseases effecting the posterior segments of the eye. In addition, the present invention is directed to particle in particle carrier formulations for sustained release of therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application Claims priority to U.S. patent application Ser. No.14/359,146, filed May 19, 2014, which is a National Stage Entry under 37U.S.C. 371 of International Application Number PCT/US2012/065620, filedNov. 16, 2012, which claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/561,256, filed Nov. 17, 2011, thedisclosure of each is included herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberEY018940 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to compounds and compositionsthereof for enhanced drug delivery, and more specifically to pro-drugderivatives of corticosteroids and non-steroid anti-inflammatory drugs(NSAIDs) and ruboxistaurin adapted for delivery to posterior segments ofthe eye. In addition, the present invention is directed to carrierformulations for sustained release of therapeutic agents.

SUMMARY OF THE INVENTION

The present invention is directed to compounds with improved retinaldelivery and efficacy and their use in treating retinal ocular disease.In addition, the present invention is directed to extended releaseformulations formed under inert conditions that maintain stability ofthe therapeutic agent while providing the desired extended releaseeffect.

In one aspect, the present invention is directed to pro-drugs ofcorticosteroids. In one exemplary embodiment, the corticosteroids arelipophilic corticosteroids that have been modified to include one ormore terminal hydrophilic acid functional group. In one exemplaryembodiment, the terminal hydrophilic acid functional group is attachedto the R at carbon 21 of the steroid backbone. Exemplary hydrophilicfunctional groups include, but are not limited to sulfates, phosphates,succinates, or salts thereof. In one exemplary embodiment, thecorticosteroid is budesonide. In another exemplary embodiment, thehydrophilic acid functional group is a sulfate functional group. Anexample of a suitable sulfate functional group salt is a sulfatetriethylammonium salt. In yet another, exemplary embodiment, thehydrophilic acid functional group is a succinate.

In another aspect, the present invention is directed to single anddouble pro-drugs of NSAIDs. The NSAIDs of the present invention may bemodified to mask basic or mildly basic terminal functional groups withone or more multi-functional acid groups. Exemplary multi-functionalacid groups include, but are not limited to, maleates, fumarates,tartates, citrates, and succinates. In one exemplary embodiment, themulti-functional acid group is a succinate. In certain exemplaryembodiment, the NSAID may be further modified to include a taurine. Thetaurine may be bound to the multi-functional acid group, or directly tothe parent molecule. In one exemplary embodiment, the NSAID is a coxib.In another exemplary embodiment, the coxib is celecoxib.

In yet another aspect, the present invention is directed to single anddouble pro-drugs of ruboxistaurin. In one exemplary embodiment, thepro-drug is a ruboxistaurin succinamidic acid. In certain exemplaryembodiments, the ruboxistaurin may be further modified to includetaurine.

In another aspect, the present invention is directed to methods oftreating retinal ocular disease using the above pro-drug compounds.Ocular diseases that may be treated with compositions of the presentinvention include ocular degenerative diseases, ocular vasculardiseases, ocular infectious diseases, and inflammatory ocular diseases.In one exemplary embodiment, the ocular disease is diabetic retinopathy.In certain exemplary embodiment, the corticosteroid pro-drug isformulated for transcleral delivery. In another exemplary embodiment,the NSAID pro-drug is formulated for topical administration to the eyein the form of eye drops.

In another aspect, the present invention is directed toparticle-in-particle (PinP) extended release compositions. The PinPcompositions comprises an inner particle which is infused within anouter particle. In one exemplary embodiment the inner particles are madefrom a material that will not expand upon exposure to a super criticalfluids (SCF), such as super critical carbon dioxide, and the outerparticle is made from a material that will expand upon exposure to SCF.Therapeutic agents may be loaded on the surface of the inner particle orouter particle, contained within the inner particle or outer particle,or contained within the pores of the outer particle, or a combinationthereof. In one exemplary embodiment, the therapeutic agent is a smallmolecule based therapeutic agent, a nucleic acid-based therapeuticagent, a viral vector, or a peptide-based agent. In another exemplaryembodiment, the therapeutic agent is a peptide-based agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of a synthetic pathway for generating budesonide21-sulfate triethylammonium salt and FIG. 1B is a schematic of asynthetic pathway for generating budesonide succinate according to anexemplary embodiment of the present invention.

FIG. 2A is a graph showing transport of budesonide, budesonide sulfateand budesonide succinate across freshly excised bovinesclera-choroid-RPE (A to B means sclera towards RPE direction.

FIG. 2B is a graph showing transport of budesonide succinate acrossfreshly excised bovine sclera-choroid-RPE in A to B and B to A direction(B to A means RPE towards sclera). Transport of this pro-drug was alsoconducted in presence of p-amino hippuric acid (a monocarboxylatetransport inhibitor) in A to B direction.

FIG. 2C is a graph showing donor amounts of budesonide succinate andbudesonide formed at time zero and at the end of the study (6 h) fortransport studies shown in panel B. Data represents mean±sd (n=4).

FIG. 3A is a schematic of a PK model for transport of budesonidesuccinate across bovine sclera-choroid-RPE in A to B direction. Circleson left side (1 BSA; 2 B) represent the donor chamber whereas circles onright side (3 BSA; 4 B) represent the receiver chamber for BSA transportin A to B direction. 2 B and 4 B were included and represent budesonideformed/transported during the course of transport study. A separatecompartment was included to account for the pro-drug/parent drug lossdue to tissue absorption. K1>0 represents rate constant for drug lossinto the tissue, K1>2 and K3>4 represent rate constants for pro-drugconversion into parent drug on donor and receiver side respectively.K1>3 and K2>4 represent the transport rate constant for budesonidesuccinate and budesonide from donor to the receiver side. K3>1 and K4>2represent transport rate constant for budesonide succinate andbudesonide from receiver to the donor side (backward movement, if any).

FIG. 3B includes graphs showing the observed and model predicted amountsof pro-drug and parent drug in donor and receiver chambers during thecourse of transport study.

FIG. 4A is a schematic showing a PK model for transport of budesonidesuccinate across bovine sclera-choroid-RPE in A to B direction inpresence of an inhibitor. Circles on left side (5 BSA; 6 B) representthe donor chamber whereas circles on right side (7 BSA; 8 B) representthe receiver chamber for BSA transport in A to B direction. 6 B and 8 Bwere included and represent budesonide formed/transported during thecourse of transport study. A separate compartment was included toaccount for the pro-drug/parent drug loss due to tissue absorption. K5>0represents rate constant for drug loss into the tissue, K5>6 and K7>8represent rate constants for pro-drug conversion into parent drug ondonor and receiver side respectively. K5>7 and K6>8 represent thetransport rate constant for budesonide succinate and budesonide fromdonor to the receiver side. K5>7 Act represent active transport rateconstant and contribution (0.5%) of active diffusion in BSA transport.K7>5 and K8>6 represent transport rate constant for budesonide succinateand budesonide from receiver to the donor side (backward movement, ifany).

FIG. 4B are graphs showing the observed and model predicted amounts ofpro-drug and parent drug in donor and receiver chambers during thecourse of transport study.

FIG. 5A is a schematic showing a PK model for transport of budesonidesuccinate across bovine sclera-choroid-RPE in B to A direction. Circleson left side (9 BSA; 10 B) represent the receiver chamber whereascircles on right side (11 BSA; 12 B) represent the donor chamber for BSAtransport in B to A direction. 12 B and 10 B were included and representbudesonide formed/transported during the course of transport study. Aseparate compartment was included to account for the pro-drug/parentdrug loss due to tissue absorption. K11>0 represents rate constant fordrug loss into the tissue, K11>12 and K9>10 represent rate constants forpro-drug conversion into parent drug on donor and receiver siderespectively. K11>9 and K12>10 represent the transport rate constant forbudesonide succinate and budesonide from donor to the receiver side.K9>11 and K10>12 represent transport rate constant for budesonidesuccinate and budesonide from receiver to the donor side (backwardmovement, if any).

FIG. 5B are graphs showing the observed and model predicted amounts ofpro-drug and parent drug in donor and receiver chambers during thecourse of transport study.

FIG. 6A is a graph showing the ex vivo transscleral delivery ofbudesonide and FIG. 6B is a graph showing the ex vivo delivery ofbudesonide succinate at the end of one hour in Brown Norway (BN) rats.Twenty-five microliters of 1 mg/ml suspension was injected into theposterior subconjunctival space of euthanized (ex vivo study) rats. FIG.6B represents ocular tissue leves of BSA and budesonide formed from BSA.Data is expressed as mean±SD for n≧3.

FIG. 7 is a graph showing a comparison of ex vivo budesonide delivery indifferent ocular tissues following posterior subconjunctival injectionof twenty-five microliters of 1 mg/ml suspension of budesonide andbudesonide succinate at the end of one hour in Brown Norwary rates. Datais expressed as mean±SD for n≧3.

FIG. 8 is a schematic of a synthetic pathway for single pro-drug(celecoxib succinamidic acid, C-SA) and double pro-drug (celecoxibsuccinamidic acid taurine, C-SA-T) of celecoxib.

FIG. 9A-C are graphs showing cumulative percentage transport (N≧4) ofcelecoxib succinamidic acid taurine (C-SA-T) double prodrug was higherthan C-SA and celecoxib across FIG. 9A cornea, FIG. 9B conjunctiva aswell as FIG. 9C sclera-choroid-RPE. Transport of double pro-drug wassignificantly reduced in the presence of an inhibitor (Taurine) acrosscornea and sclera-choroid-RPE. Data represents mean±s.d.

FIG. 10A-C are graphs showing isothermal titration calorimetry (ITC)thermograms representing the binding of FIG. 10A celecoxib (2 μg/ml);FIG. 10B C-SA (single pro-drug, 250 μg/ml); and FIG. 10C C-SA-T (doublepro-drug, 250 μg/ml) with 1 mg/ml suspension of melanin in PBS (pH 7.4).Melanin binding of C-SA-T (K=7.93 E3 M⁻¹) and C-SA (K=8.89 E3 M⁻¹) was˜130-110 fold lower than that of celecoxib (K=1.01 E6 M⁻¹).

FIG. 11 A-D are graphs showing ocular biodistribution at the end of 1 hafter administration of 10 μl drop of FIG. 11A celecoxib (2 mg/ml), FIG.11B C-SA (2 mg/ml), FIG. 11C C-SA-T (2 mg/ml), and FIG. 11D C-SA-T (10mg/ml) in BN rats. Data represents mean±sd (n=6-8 eyes, 3-4 animals).Panels B, C, D represent the levels of pro-drug as well as parent drugformed.

FIG. 12 is a graph showing ocular biodistribution of celecoxib at theend of 1 h after administration of 10 μl drop of celecoxib (2 mg/ml),C-SA (2 mg/ml), C-SA-T (2 mg/ml), and C-SA-T (10 mg/ml) in BN rats. Datarepresents mean±sd (n=6-8 eyes, 3-4 animals).

FIG. 13A-C are graph showing the ability of eye drops of 1% w/vcelecoxib succinamidic acid-taurine was effective in reducing the bloodretinal barrier leakage at the end of 2 months treatment in STZ-induceddiabetic animals as assessed by FIG. 13A FITC-Dextran (4 Kda) leakageassay, FIG. 13B Vitreous to plasma protein ratio; and FIG. 13Cleukostasis. Data represents mean±sd (n=6-8 eyes, 3-4 animals; *Significantly different from diabetic+Vehicle group, Student's t-test,p<0.05).

FIG. 14 is a schematic showing loading of SCF treated PLGAmicroparticles with bevacizumab and the cumulative release rate ofbevacizumab from those microparticles.

FIG. 15 is a schematic showing an alternative process for loading PLGAmicroparticles with bevacizumab using SCF and the corresponding releaserate of bevacizumab from those particles.

FIG. 16 is schematic showing the initial loading of bevacizumab on PLAnanoparticles following by nanoparticle infusion into and expansion ofPLGA microparticles using SCF according to an exemplary embodiment ofthe present invention and the resulting extended release profiles ofbevacizumab from those particles.

FIGS. 17A-H re a series of confocal micrographs at increasing resolutionshowing the surface areas of PLGA microparticles before and aftertreatment with SCF.

FIG. 18 is a panel of confocal micrographs showing the infusion of PLGAmicroparticles with PLA nanoparticles after exposure to SCF.

FIG. 19 is a graph showing the cumalitve in vitro release rate ofbevacizumab from exemplary particle in particle (PinP) compositions.

FIG. 20 is a graph showing the circular dichorism (CD) spectra of nativebevacizumab, supercritical treated CO₂ bevacizumab, and bevacizumab fromin vitro release samples (1, 2, 3, and 4 months).

FIG. 21 is a size exclusion chromatogram of native, supercritical CO₂treated bevacizumab and in vitro release of bevacizumab from exemplaryPinP compositions.

FIGS. 22A-C are scanning electron microscopy pictures of PLAnanoparticles infused into porous PLGA microparticles (NPinPMP) atvarious magnifications.

FIGS. 23A and B are pictures of SGS PAGE gels demonstrating the resultsof a stability evaluation of native bevacizumab, supercritical treatedCO₂ bevacizumab, and bevacizumab from in vitro release samples after 1,2, 3, and 4 months. FIG. 23A represents a reducing gel. FIG. 23Brepresents a non-reducing gel.

FIGS. 24A-C are graphs depicting the results of non-invasive ocularfluorophotometry of rat eyes after intravitreal injection ofAlexa-bevacizumab solution (FIG. 24A) and Alexa-bevacizumab loaded in anexemplay PinP composition (FIG. 24B). FIG. 24C the fluorescence levelsof Alexa-bevacizumab alone and Alexa-bevacizumab in PiP up to 60 dayspost-delivery.

FIG. 25 is a graph showing the cumaltive release of His-LEDG₁₋₃₂₆fromexemplary PinP compositions.

FIG. 26A-C are graphs showing the results of non-invasive ocularfluorophotometry after intravitreal injection in rat eyes ofAlexa-His-LEDGF₁₋₃₂₆ solution (FIG. 26A) and Alexa-His-LEDGF₁₋₃₂₆ loadedin PinP (FIG. 26B). FIG. 26C indicates the His-LEDG₁₋₃₂₆concentrationsfor the PinP and solution injected groups.

DETAILED DESCRIPTION

As used herein “retina and retinal” refers both to the retina as well asthe general posterior segment of the eye adjacent to the retina.

As used herein “treating or treatment” refers to a complete reversal orelimination of the underlying disease, a temporary or sustainedprevention of disease progression, a temporary or sustained regressionof the disease, and amelioration of one or more symptoms associated withthe disease.

There are a number of ocular diseases that affect posterior segments ofthe eye, including the retina. Treatment of diseases affecting theposterior segment of the eye have typically included topical, systemicand intravitreal administration of therapeutic agents. Therapeuticagents administered topically must traverse the cornea, lens, trabecularnetwork, and blood-aqueous barrier to reach the retina, with the netresult that very little therapeutic agent generally reaches theposterior segment of the eye. Systemic administration requires traversalof the blood-retinal-barrier often requiring high doses that can lead tounwanted side effects. Intravitreal administration is highly invasiveand carries the risks of retinal detachment, endophthalmitis andcataract. Accordingly, ideal therapeutic agents would not only exert thedesired pharmacological effect, but traverse the unique ocular barriersof the eye and exert a localized effect at the site of disease.

A number of corticosteroids have shown promise in treating retinaldiseases. However, corticosteroids have been limited by poor solubilityand permeability profiles across the back of the eye. Accordingly, inone aspect, the present invention is directed to pro-drugs ofcorticosteroids with enhanced retinal delivery profiles and their use intreating retinal ocular diseases. In addition, a number of NSAIDs alsopossess pharmacological effects useful in treating ocular diseases, butas with corticosteroids, suffer from poor solubility, limitedpermeability of the eye, or a combination thereof. Accordingly, inanother aspect, the present invention is directed to single and doublepro-drugs of NSAIDs and their use in treating retinal ocular disease.The double prodrug concept can be applied to corticosteroids as well asother drugs. Further, it is often desirable to obtain sustained orextended delivery when delivering therapeutic agents, including thosedescribed above. When formulating therapeutic agents for extendeddelivery it is often necessary to rely upon a carrier that can controlthe rate of release of the therapeutic agent. Direct loading of atherapeutic agent on a carrier may fail to exert the desired or optimalextended release effect. Likewise, encapsulation of certain therapeuticagents in a carrier can adversely affect the therapeutic agentsstability as a result of the reaction conditions necessary toencapsulate the therapeutic agent in the carrier. Accordingly, inanother aspect, the present invention is directed to extended releaseformulations that allow therapeutic agents to be loaded within extendedrelease carriers under inert conditions.

Corticosteroid Pro-drugs

Corticosteroids are a group of natural and synthetic analogues of thehormones secreted by the hypothalamic-anterior pituitary-adrenocortical(HPA) axis. These include glucocorticoids, mineralcorticoids, andcorticotropins. The chemical modifications of the present invention maybe used to improve the solubility and dissolution rates ofcorticosteroids. Suitable corticosteroids for use in the presentinvention can be selected, for example, by their ability to demonstratean anti-inflammatory effect as well as other useful pharmacologicaleffects (e.g. anti-angiogenic) in treating ocular diseases by using invivo retinal cell models. Exemplary corticosteroids of the presentinvention include, but are not limited to, triamcinolone, prednisolone,dexamethasone, fluocinolone acetonide, triamcinlone acetonide,hydrocortisone, methyprednisolone, betamethasone, beclomethasone,fludrocortisones, prednisone, and budesonide. By way of example, but notof limitation, budesonide is in clinical use for treatment of asthma,allergic rhinitis, and inflammatory bowel disease. It is a potentanti-inflammatory corticosteroid with a 1000 fold higher topicalanti-inflammatory effect than cortisol. (1, 2). It is capable ofreducing vascular epithelial grown factor (VEGF) secretion and mRNAexpression in retinal pigment epithelia cells (ARPE19 cells) viaglucocorticoid-receptor mediated mechanisms at nanomolar concentrations.Accordingly, budesonide is representative of a type of corticosteroidthat may be useful in the context of the present invention for treatingretinal ocular diseases.

The corticosteroid pro-drugs of the present invention containmodifications which increase the hydrophilicity of the corticosteroid. Apro-drug is generated by the attachment of one or more functional groupsto the steroid backbone. The functional groups may be attached to the A,B, C, or D rings of the parent steroid backbone or to functional groupsexisting on the active corticosteroid. In one exemplary embodiment, thecorticosteroid includes the introduction of at least one negativelycharged terminal group to the parent compound. While not bound by thefollowing theory, it is believed that the negatively charged terminalgroups reduce binding of the corticosteroid to melanin and or othernative eye components that result in the sequestration or removal of thecorticosteroid from the eye. The negatively charged terminal acid may bebound to the parent compound by in vivo hydrolysable esters, amides,carbamates or other acceptable pro-drug linkages. In one exemplaryembodiment, the functional groups are attached to the corticosteroid viaester linkages. In certain exemplary embodiments, the pro-drugfunctional group is attached to a terminal hydroxide present on the Dring or to an existing functional group attached thereto. In anotherexemplary embodiment, the pro-drug functional group is attached to aterminal hydroxy on a functional group attached to carbon 17 of thecorticosteroid backbone. Functional groups that may be used to generatethe corticosteroid pro-drugs of the present invention includedsulphates, sulfphones, sulfoxides, sulphonic acids, citrates,phosphates, phosphines, phosphodiesters, phosphonic acids succinates, orsalts thereof. In addition, functional groups that may be used in thepresent invention include the follower esters; maleate, citrate,tartrate, adipic acid, glutaric acid, malonic acid, and hydroxy succinicacid, hydroxy succinic acid esters. In one exemplary embodiment, thefunctional group is a sulphate or sulphate salt. In certain exemplaryembodiments, the sulfate salt is an ammonium salt, such as, a sulfatetriethylammonium salt. In another exemplary embodiment, the pro-drugfunctional group is a succinate or acid thereof.

In one exemplary embodiment, pro-drugs of the present invention have thefollowing general formula:

wherein R is any one of the R-groups listed in Table 1 below.

TABLE 1 Physicochemical properties of budesonide and prodrugs thereofPredicted Solute Mol. Predicted¹ Predicted¹ net mol. Sl. (Sol_(aq);mg/ml; Mol. Radius Log D Predicted¹ pK_(a1), pK_(a2) charge at No pH7.4; 25° C.) —R group Wt. (nm) (pH 7.4) Log P (acidic) pH 7.4 15Budesonide (0.02) —H 430.5 0.55 1.81 1.81 14.4, 15.7  0   16 Adipic acidmono- —COCH₂CH₂CH₂CH₂COOH 558.7 0.60 0.4 2.71 4.6, 16.0 1− budesonideester 17 Glutaric acid mono- —COCH₂CH₂CH₂COOH 544.5 0.60 −0.15 2.37 4.5,16.0 1− budesonide ester 18 Succinic acid mono- —COCH₂CH₂COOH 530.6 0.59−0.51 2.26  4.2, 16.05 1− budesonide ester 19 Malonic acid mono-—COCH₂COOH 516.5 0.59 −1.71 1.92 3.1, 15.6 1− budesonide ester 20Hydroxy succinic acid —COCH₂CH(OH)COOH 546.5 0.60 −2.0 1.75 3, 16, 16.11− mono-budesonide ester 21 Budesonide phosphate —PO(OH)₂ 510.5 0.59−3.7 1.04 1, 6.2, 16 2−

Pro-drugs of NSAIDs

NSAIDs are a class of drugs which includes members havingeicosanoid-depressing and anti-inflammatory properties. Likecorticosteroids members of the class can suffer from low solubility andpermeability of the eye. NSAIDs possess a number of pharmacologicalproperties that may be useful in treating ocular diseases By way ofexample, but not of limitation, celecoxib is a potent and selectiveCOX-2 inhibitor for use in the treatment of inflammatory diseases likerheumatoid arthritis and osteoarthritis. It has been shown previouslythat levels of prostaglandins increase in the diabetic retinas via COX-2mediated processes (3,4). In addition, the levels of vascularendothelial growth factor (VEGF) mRNA and COX-2 expression in retinalpigment epithelial cells increase early in the course of diabeticretinopathy (5). Increased production of prostaglandins can alsostimulate VEGF production in the retina (6). An increase inprostaglandin E2 (PGE2) and VEGF levels results in the breakdown of theblood-retinal-barrier leading to vascular linkage (7-9). Accordingly,NSAIDs with properties such as, but not limited to those of celecoxib,represent suitable NSAIDs for use in the context of the presentinvention for treatment of retinal ocular diseases. In one exemplaryembodiment, the coxib is celecoxib. NSAIDs that may be used in thepresent invention include salicylates, propionic acid derivatives,acetic acid derivatives, enolic acid (oxicam) derivatives, fenamic acidderivatives (fenamates) and selective COX-2 inhibitors (coxibs). In oneexemplary embodiment, the NSAID is a coxib. Exemplary coxibs include,but are not limited to, celecoxib, rofecoxib, valdecoxib, parecoxib,lumiracoxib, etoricoxib, and firocoxib. In one exemplary embodiment, thecoxib is celecoxib. In another exemplary embodiment, the NSAID isdiclofenac, ketoralac, nepafenac, or bromfenac. In another exemplaryembodiment the COX-2 inhibitor is nimesulide.

The present invention comprises single and double pro-drugs of NSAIDs.The pro-drugs of the present invention comprise a NSAID with a terminalfunctional group containing a weak base to which a multi-functionalcounter acid is bound. In one exemplary embodiment, the weak base on theparent compound has a pKa value of approximately 7 to approximately 10.The multi-functional counter acid may be bound to the parent compound byin vivo hydrolysable esters, amides, carbamates or other acceptablepro-drug linkages. In one exemplary embodiment, the counter-acid isbound by an in vivo hydrolysable ester. While not bound by the followingtheory, it is believed that functional groups containing basic groupsincrease melanin binding. Melanin binding acts as a major barrier toeffective distribution of a compound in an eye by sequestering orotherwise preventing the compound from reaching its target. Accordingly,modifications to the parent NSAID compound that can reduce melaninbinding are contemplated by the present invention. As used herein, “amulti-functional compound or counter acid” is a molecule containingmultiple functional groups. For example a multi-functional group can bea compound with a carbon backbone to which multiple functional groupsare attached or single molecules such as phosphates and sulfates.Suitable multi-functional counter acids for use in the present inventioninclude, but are not limited to, aspartates, maleates, fumarates,tartarates, citrates, amides and succinates. Exemplary amides that maybe used in the present invention include ethyl butyramide,privaloylamide, butyamide, propionamide, acetamide, sinapamide,salicylamide, a succinamide, sinapamide, and a gycinamide. In oneexemplary embodiment, the multi-functional counter ion is a succinate oracid thereof.

In one exemplary embodiment the pro-drug of the present invention is acelecoxib modified to mask a terminal sulfonamide group (predicted pKavalue of 8.8) with a succinic anhydride to form a celecoxib succinamidicacid. A synthetic pathway for generating a celecoxib succinamidic acidis shown in FIG. 8 and described in further detail in the Examplessection below.

In another exemplary embodiment, the pro-drug of the present inventionhas the following general formula:

wherein R is any one of the R-groups listed in Table 2 below.

TABLE 2 Physicochemical properties of celecoxib/prodrugs selected fortransscleral transport studies Predicted Solute Mol. Predicted¹Predicted¹ net mol. Sl. (Sol_(aq); mg/ml; Mol. Radius Log D Predicted¹pK_(a1), pK_(a2) charge at No pH 7.4; 25° C.) —R group Wt. (nm) (pH 7.4)Log P (acidic) pH 7.4 1 Celecoxib (0.005) —H 381 0.53 3.32 3.35 8.8, 8.80   2 Methyl-ethanolamine mannich —CH₂N(CH₃)CH₂CH₂OH 468 0.57 3.01 3.088.57 0   base of celecoxib 3 Celecoxib ethyl butyramide —COCH(CH₂CH₃)₂479 0.57 2.5 4.4 5.04 0   4 Celecoxib pivaloylamide —COC(CH₃)₂ 465 0.571.9 3.8 5.04 0   5 Celecoxib butyramide —COCH₂CH₂CH₃ 463 0.57 1.7 3.954.8  0   6 Celecoxib propionamide —COCH₂CH₃ 449 0.57 1.5 3.75 4.74 0   7Celecoxib-acetamide —COCH₃ 423 0.55 1.2 2.98 5.0  0   8 Celecoxibsinapamide —COCH═CHC₆H₂(4-OH)2,5- 587 0.62 1.0 3.0 5.04, 8.49 1− (OCH₃)₂9 Celecoxib 4-amino salicylamide —COC₆H₃(OH)(NH₂) 516 0.59 0.75 2.765.04, 9.08 1+ 10 Celecoxib —COC(CH₂OH)₂CH₂CH₃ 512 0.59 0.6 2.6 5.04,13.9 0   bis(hydroxymethyl)butyramide 11 Celecoxib octenyl succinamidic—COCH₂CH(C₈H₁₅)COOH 605 0.63 −0.04 4.8 4.4, 4.8 1− acid 12 Celecoxibgycinamide —COCH₂NH₂ 438.5 0.56 −0.4 2.1 4.74 1+ 13 Celecoxib adipamidicacid —COCH₂CH₂CH₂CH₂COOH 509 0.59 −1.1 3.1 4.73, 5.04 1− 14 Celecoxibglutaramidic acid —COCH₂CH₂CH₂COOH 495 0.58 −1.95 2.76 4.54, 4.74 1− 15Celecoxib succinamidic acid —COCH₂CH₂COOH 481 0.57 −4.8 2.53 4.4, 5.0 1−(1.0) 16 Celecoxib-maleiamidic acid —COCH═CHCOOH 479 0.57 −6.1 2.6 2.3,4.3, 4.7 1− 17 Celecoxib malonamidic acid —COCH₂COOH 467 0.57 −3.86 2.42.98, 4.74 1− 18 Celecoxib ethyl succinamide —COCH₂CH₂COOCH₂CH₃ 509 0.591.34 3.6 4.74 0   19 Celecoxib ethyl maleiamide —COCH═CHCOOCH₂CH₃ 5070.59 1.45 3.71 4.74 0   20 Celecoxib-N,N-dimethyl- —COCH(N(CH₃)₂)CH₃COOH524 0.59 −2.84 3.06 2.7, 5.0 1− aspartamidic acid 21Celecoxib-ethoxysuccinamidic —COCH₂CH(OCH₂CH₃)COOH 525 0.59 −3.33 2.623.8, 5.0 1− acid 22 Celecoxib-hydroxysuccinamidic —COCH₂CH(OH)COOH 4970.58 −3.82 2.06 3.9, 5.0 1− acid 23 Celecoxib-aspartamidic acid—COCH(NH₂)CH₂COOH 496 0.58 −3.97 1.63 3.3, 5.0 0  

In yet another exemplary embodiment, the pro-drug of the presentinvention has the following general formula:

where R is one of the R-groups listed in Table 3 below.

TABLE 3 Physicochemical properties of nimesulide and prodrugs thereofPredicted Solute Mol. Predicted¹ Predicted¹ net mol. Sl. (Sol_(aq);mg/ml; Mol. Radius Log D Predicted¹ pK_(a1), pK_(a2) charge at No pH7.4; 25° C.) —R group Wt. (nm) (pH 7.4) Log P (acidic) pH 7.4 1Nimesulide (0.007) —H 308.3 0.50 3.8 3.8 8.5  0   2 Nimesulide acetamide—COCH₃ 350.9 0.52 2.36 2.36 none 0   3 Nimesulide glycinamide —COCH₂NH₂365.07 0.52 1.16 1.16 6.52 1+ 4 Nimesulide adipamidic acid—COCH₂CH₂CH₂CH₂COOH 436.4 0.56 −0.22 2.0 4.73 1− 5 Nimesulideglutaramidic —COCH₂CH₂CH₂COOH 422 0.55 −0.7 2.26 4.5  1− acid 6Nimesulide N,N-dimethyl —COCHN(CH₃)₂CH₂COOH 451.5 0.56 −1.7 1.32 2.71 1−aspartamidic acid 7 Nimesulide malonamidic —COCH₂COOH 394.5 0.54 −2.461.52 2.06 1− acid 8 Nimesulide succinamidic —COCH₂CH₂COOH 408.4 0.54−4.3 1.4 4.04, 4.15 1− acid

In certain exemplary embodiments, the NSAID pro-drugs of the presentinvention further comprise the addition of a taurine to form a doublepro-dug compound. Taurine is accumulated into photoreceptor cells and toa certain extent Muller, amacrine and bipolar cells (10-13). Inaddition, taurine has been reported to penetrate through theblood-retinal-barrier into the retina. Supplementation with taurine hasbeen documented to reduce the up-regulation of VEGF expression in astreptozotocin-induced diabetic retinopathy rat model (14). The zwitterionic nature of taurine is believed to hinder its passive diffusionthrough lipid membranes. The observed retinal influx clearance oftaurine was found to be significantly higher than those of non-permeableparacellular markers, indicating a carrier-mediated rather thandiffusion mediated process.

In one exemplary embodiment, the taurine is bound to a terminalfunctional group on the multi-functional counter acid. In anotherexemplary embodiment, the taurine is bound directly to the NSAID parentcompound. In yet another exemplary embodiment, more than one taurine maybe bound to the multi-functional counter acid, the parent compound, or acombination thereof. Similar concepts can be applied to other drugsincluding corticosteroids. In one exemplary embodiment taurine is boundto a celcoxib pro-drug of the present invention. In another exemplaryembodiment, the taurine is bound to a celecoxib succinamidic acidpro-drug. A synthetic pathway for binding taurine to a celecoxibsuccinamidic acid is shown in FIG. 8 and described in further detail inthe Examples section below.

Ruboxistaurin Pro-Drugs

The present invention comprises single and double pro-drugs ofruboxistaurin. Ruboxistaurin is a protein kinase C beta inhibitor theoverexpression of which has been implicated in the development ofdiabetic retinopathy. In one exemplary embodiment, the pro-drug of thepresent invention has the following general formula:

wherein R is aspartates, maleates, fumarates, tartarates, citrates,amides, succinates, or ester or amidic acids thereof. In one exemplaryembodiment, R is any one of the R-groups listed in Table 4 below.

TABLE 4 Physicochemical properties of p ruboxistaurin and prodrugsthereof Predicted Solute Mol. Predicted¹ Predicted¹ net mol. Sl.(Sol_(aq); mg/ml; Mol. Radius Log D Predicted¹ pK_(a1), pK_(a2) chargeat No pH 7.4; 25° C.) —R group Wt (nm) (pH 7.4) Log P (acidic) pH 7.4 9Ruboxistaurin (0.029) —H 468.5 0.57 2.2 3.9 8.26 0   10 Ruboxistaurinacetamide —COCH₃ 512.66 0.59 1.7 3.2 none 0   11 Ruboxistaurinadipamidic —COCH₂CH₂CH₂CH₂COOH 582.7 0.61 0.35 3.1 4.7  1− acid 12Ruboxistaurin glutaramidic —COCH₂CH₂CH₂COOH 568 0.60 −0.9 2.21 4.57 1−acid 13 Ruboxistaurin malonamidic —COCH₂COOH 540 0.60 −1.25 1.77 2.45 1−acid 14 Ruboxistaurin succinamidic —COCH₂CH₂COOH 554.6 0.60 −2.8 2.84.21, 4.31 1− acid

In certain exemplary embodiments, the ruboxistaurin pro-drugs of thepresent invention further comprise the addition of a taurine to form adouble pro-dug compound. In one exemplary embodiment, the taurine isbound to a terminal functional group on the R-group. In anotherexemplary embodiment, the taurine is bound directly to the ruboxistaurinparent compound. In yet another exemplary embodiment, more than onetaurine may be bound to the R-group, the parent compound, or acombination thereof.

Pharmaceutical Compositions

The corticosteroid and NSAID pro-drug compounds described herein can beprovided as physiologically acceptable formulations using knowntechniques, and the formulations can be administered by standard routesincluding but not limited to topical, periocular or transscleral,suprachoroidal, subretinal, intravitreal and systemic routes. 100% pureisomers are contemplated by this invention; however a stereochemicalisomer (labeled as α or β, or as R or S) may be a mixture of both in anyratio, where it is chemically possible by one skilled in the art. Alsocontemplated by this invention are both classical and non-classicalbioisosteric atom and substituent replacements, such as are described byPatani and Lavoie (“Bio-isosterism: a rational approach in drug design”Chem. Rev. (1996) p. 3147-3176) and are well known to one skilled in theart. Such bioisosteric replacements include, for example, but are notlimited to, substitution of a S or a NH for an O.

The formulations in accordance with the present invention can beadministered in the form of a tablet, a capsule, a lozenge, a cachet, asolution, a suspension, an emulsion, a powder, an aerosol, asuppository, a spray, a pastille, an ointment, a cream, a paste, a foam,a gel, a tampon, a pessary, a granule, a bolus, a mouthwash, an eye dropor a transdermal patch.

The formulations include those suitable for oral, rectal, nasal,inhalation, topical (including dermal, transdermal, buccal, and eyedrops), vaginal, parenteral (including subcutaneous, intramuscular,intravenous, intradermal, intraocular (including local injections suchperiocular, suprachoroidal, subretinal, and intravitreal),intratracheal, and epidural) or inhalation administration. In oneexemplary embodiment, the corticosteroid pro-drugs of the presentinvention are formulated for transcleral delivery. Transcleral deliveryincludes subconjunctival, subtenons', and retrobulbar trancleraldelivery. In one exemplary embodiment, the NSAID pro-drugs areformulated for administration topically as eye drops. The formulationscan conveniently be presented in unit dosage form and can be prepared byconventional pharmaceutical techniques. Such techniques include the stepof bringing into association the active ingredient and a pharmaceuticalcarrier(s) or excipient(s). In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored base, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert base such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels, pastes, and eye drops comprisingthe ingredient to be administered in a pharmaceutical acceptablecarrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken; i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining, in addition to the active ingredient, ingredients such ascarriers as are known in the art to be appropriate.

Formulation suitable for inhalation may be presented as mists, dusts,powders or spray formulations containing, in addition to the activeingredient, ingredients such as carriers as are known in the art to beappropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. Formulations suitable for parenteral administration alsoinclude, but are not limited to, nanoparticle formulations made bynumerous methods as disclosed in U.S. patent application Ser. No.10/392,403 (Publication No. US 2004/0033267), U.S. patent applicationSer. No. 10/412,669 (Publication No. US 2003/0219490), U.S. Pat. No.5,494,683, U.S. patent application Ser. No. 10/878,623 (Publication No.US 2005/0008707), U.S. Pat. No. 5,510,118, U.S. Pat. No. 5,524,270, U.S.Pat. No. 5,145,684, U.S. Pat. No. 5,399,363, U.S. Pat. No. 5,518,187,U.S. Pat. No. 5,862,999, U.S. Pat. No. 5,718,388, and U.S. Pat. No.6,267,989, all of which are hereby incorporated herein by reference inthere entirety. A review of drug formulation technology is provided in“Water Insoluble Drug Formulation” by Rong Liu, editor, pp. 1-633,(2000) CRC Press LLC, which is incorporated herein by reference in itsentirety.

It should be understood that, in addition to the ingredientsparticularly mentioned above, the formulations of the present inventionmay include other agents conventional in the art having regard to thetype of formulation in question, for example, those suitable for oraladministration may include flavoring agents, and nanoparticleformulations (e.g.; less than 2000 nanometers, preferably less than 1000nanometers, most preferably less than 500 nanometers in average crosssection) may include one or more than one excipient chosen to preventparticle agglomeration.

Retinal Ocular Diseases and Methods of Use

The corticosteroid, NSAID, and ruboxistaurin single and double pro-drugformulations of the present invention may be used to treat diseaseseffecting the posterior segment of the eye. For ease of reference thesingle and double pro-drug formulations of the present invention arereferred to collectively below as the pro-drug formulation. Diseasesaffecting the posterior segment of the eye that may be treated with thecorticosteroid and NSAID pro-drug compounds of the present inventioninclude degenerative, vascular, inflammatory, and infectious diseasesaffecting the posterior segment of the eye. Exemplary degenerativediseases include, but are not limited to, ARMD, and retinitispigmentosa. Exemplary vascular diseases include, but are not limited to,diabetic retinopathy and choroidal neovascularization. Exemplaryinflammatory diseases include, but are not limited to uveitis. Exemplaryinfectious diseases include, but are not limited to CMV retinitis. Inaddition, the corticosteroid and NSAID pro-drug compounds of the presentinvention may used to treat glaucoma and optic neuritis.

In one exemplary embodiment, the present invention comprisesadministering to a patient with a disease affecting the posteriorsegment of the eye a composition comprising a corticosteroid pro-drug ofthe present invention, a NSAID pro-drug of the present invention, aruboxistaurin pro-drug or a combination thereof. In one exemplaryembodiment the corticosteroid pro-drug is delivered transclerally. Inanother exemplary, embodiment, the NSAID pro-drug is administered to theeye topically in the form of eye drops. In another exemplary embodiment,the corticosteroid pro-drug, NSAID pro-drug, ruboxistaurin pro-drug or acombination thereof are implanted or systemically administered in anextended release formulation. In one exemplary embodiment, the extendedrelease formulation is a nanoparticle in porous microparticle (NPinPMP)formulation according to the present invention and described in greaterdetail below.

In another exemplary embodiment, the present invention comprises methodsreducing blood retinal barrier leakage comprising administering to apatient in need thereof a composition comprising a corticosteroidpro-drug of the present invention, a NSAID pro-drug, a ruboxistaurinpro-drug of the present invention, or a combination thereof. In oneexemplary embodiment the corticosteroid pro-drug is deliveredtransclerally. In another exemplary, embodiment, the NSAID pro-drug isadministered to the eye topically in the form of eye drops. In anotherexemplary embodiment, the corticosteroid pro-drug, NSAID pro-drug,ruboxistaurin pro-drug or a combination of both are implanted orsystemically administered in an extended release formulation. In oneexemplary embodiment, the extended release formulation is a nanoparticlein porous microparticle formulation according to the present inventionand described in greater detail below.

In another exemplary embodiment, the present invention comprises methodsof reducing retinal leukostasis comprising administering to a patient inneed thereof a composition comprising a corticosteroid pro-drug of thepresent invention, a NSAID pro-drug of the present invention, aruboxistaurin pro-drug, or a combination thereof. In one exemplaryembodiment the corticosteroid pro-drug is delivered transclerally. Inanother exemplary, embodiment, the NSAID pro-drug is administered to theeye topically in the form of eye drops. In another exemplary embodiment,the corticosteroid pro-drug, NSAID pro-drug, or a combination of bothare implanted or systemically administered in an extended releaseformulation. In one exemplary embodiment, the extended releaseformulation is a nanoparticle in porous microparticle formulationaccording to the present invention and described in greater detailbelow.

In another exemplary embodiment, the present invention comprises methodsof reducing PGE2 levels in a posterior segment of the eye comprisingadministering to a patient in need thereof a composition comprising acorticosteroid pro-drug of the present invention, a NSAID pro-drug ofthe present invention, a ruboxistaurin pro-drug, or a combinationthereof. In one exemplary embodiment the corticosteroid pro-drug isdelivered transclerally. In another exemplary, embodiment, the NSAIDpro-drug is administered to the eye topically in the form of eye drops.In another exemplary embodiment, the corticosteroid pro-drug, NSAIDpro-drug, ruboxistaurin pro-drug or a combination of both are implantedor systemically administered in an extended release formulation. In oneexemplary embodiment, the extended release formulation is a nanoparticlein porous microparticle formulation according to the present inventionand described in greater detail below. As used herein reduction of PGE2levels includes a reduction in regulatory components involved in thebiosynthesis of PGE2 levels as well as physical PGE2 levels itself. Areduction in PGE2 levels is based on levels bellowed those observed incomparable healthy tissues. In one exemplary embodiment, methods of thepresent invention result in a reduction of at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, %50, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of PGE2 levels below that observed in comparablehealthy tissue.

In another exemplary embodiment, the present invention comprises methodsof reducing VEGF levels in a posterior segment of the eye comprisingadministering to a patient in need thereof a composition comprising acorticosteroid pro-drug of the present invention, a NSAID pro-drug ofthe present invention, a ruboxistaurin pro-drug, or a combinationthereof. In one exemplary embodiment the corticosteroid pro-drug isdelivered transclerally. In another exemplary, embodiment, the NSAIDpro-drug is administered to the eye topically in the form of eye drops.In another exemplary embodiment, the corticosteroid pro-drug, NSAIDpro-drug, ruboxistaurin pro-drug or a combination of both are implantedor systemically administered in an extended release formulation. In oneexemplary embodiment, the extended release formulation is a nanoparticlein porous microparticle formulation according to the present inventionand described in greater detail below. As used herein reduction of VEGFlevels includes a reduction mRNA and/or protein levels of VEGF as wellas in regulatory components involved in the transcription andtranslation of VEGF. A reduction in VEGF levels is based on levelsbellowed those observed in comparable healthy tissues. In one exemplaryembodiment, methods of the present invention result in a reduction of atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, %50, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of PGE2 levels below thatobserved in comparable healthy tissue.

Particle-in-Particle Extended Release Formulations

In another aspect the present invention is directed toparticle-in-particle (PinP) extended release compositions. The extendedrelease compositions of the present invention comprise an inner particlecontained within a larger porous outer particle, including variousarchitectures such as a nanoparticle in porous microparticle (NPinPMP),small nanoparticle in porous large nanoparticle (SNPinPLNP), and smallmicroparticle in porous large microparticle (SMPinPLMP). The innerparticle is smaller and relatively non-expandable as compared to thelarger outer particle during processing. The outer particle isexpandable and forms a significantly porous structure during processingthat allows the embedding of the inner particle within the outerparticle's porous structure.

As used in the context of the present invention, a particle isconsidered to expand in the presence of a supercritical fluid if theparticle's initial surface area increases within a range ofapproximately 1.25 to approximately 100 times. In certain exemplaryembodiments, the particle is considered to expand if the particle'sinitial surface surface area expands within a range of approximately1.25 to approximately 5 times, approximately 5 to approximately 25times, approximately 25 to approximately 50 times, approximately 50 toapproximately 75 times, or approximately 75 to 100 times. Alternatively,a particle is considered to expand if the particle's initial sizeincreases by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50%.

Inner particles of the present invention are made using polymeric ornon-polymeric materials that do not expand in the presence of asupercritical fluid. In certain exemplary embodiments, the nanoparticlematerial is a polymeric material that will not expand in the presence ofsupercritical fluids. In certain exemplary embodiments, the polymericmaterial is a material that will not expand in the presence ofsupercritical carbon dioxide. Examples of suitable polymeric andnon-polymeric materials that may be used in the present inventioninclude polylactide (PLA), poly(glycolic acid), co-polymers of lacticand glycolic acid (PLGA), cellulose derivatives, chitosan, polyethylene(PE), polypropylene, poly(tetrafluoroethylene), poly(ethyleneterephathalate), iron oxide, cerium oxide, zinc oxide, gold, silver,other biocompatible metals and crystals, and silica. Crystallinematerials or those with large crystalline regions are less likely toexpand during supercritical fluid processing. Polymeric inner particlesmay be prepared using conventional emulsion-solvent evaporation methodsor other similarly suitable synthesis methods. Therapeutic agents may beencapsulated in the inner particles during formation or loaded on thesurface after formation of the inner particles.

Outer particles of the present invention are made using materials thatexpand in the presence of a supercritical fluid. In certain exemplaryembodiments, the microparticle material is a polymeric material thatexpands in the presence of a supercritical fluid. In certain exemplaryembodiments, the material that expands in the presence of supercriticalcarbon dioxide. Examples of suitable polymeric materials that may beused in the present invention include lactide-co-glycolide, polyamides,polycarbonates, polyakylene glycols, polyalkylene oxides, polyvinylalcohols, polyvinyl ethers, polyvinly esters, polyvinylpyrrolidone,polyglycolides, and co-polymers thereof. In addition, sutiable polymermaterials also include alkyl cellulose, hydroxyalkyl celluloses,cellulose ethers, cellulose esters, nitro celluloses, polymers ofacrylic and methacrylic esters, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose acetate butyrate,cellulose acetate phthalate, carboxylethyl cellulose, cellulosepoly(methyl methacrylate), poly(elthylmethacrylate),poly(butymethacrylate), poly(vinyl alcohols), poly(vinyl acetate), andpolyvinylpryrrolidone. In general, amorphous materials or those withlarge amorphous regions are suitable for expansion during supercriticalfluid processing. Polymeric outer particles may be prepared usingconventional emulsion-solvent evaporation, or other similarly suitablesynthesis methods. In certain exemplary embodiments, therapeutic agentsmay be encapsulated in the outer particles during formation or loaded onthe surface after formation of the outer particles.

The extended release compositions may be used to deliver a wide range oftherapeutic agents where an extended release profile is desired. Theprocess of generating various particle architectures is achieved usingsupercritical fluid flow technology. The resulting organic solvent-freeloading is especially well suited to drugs, such as peptide andnucleotide based drugs, and viral vectors, which are susceptible toaggregation or degradation. A therapeutic agent may be loaded on thesurface of the inner particle, the outer particle or both; in the matrixof the inner particle, outer particle or both; present in the pores ofthe outer particle; or a combination thereof. In certain exemplaryembodiments, therapeutic agents may be present on the surface of theinner particle. In another exemplary embodiment, therapeutic agents maybe present on the surface of the inner and outer particle. In yetanother exemplary embodiment, therapeutic agents may be present in thematrix of the inner particle. In another exemplary embodiment, atherapeutic agent may be present in the matrix of both the inner andouter particle. In another exemplary embodiment, a therapeutic agent mayfurther be present in the porous structure of the outer particle.

Therapeutic agents that may be loaded on the nanoparticles includesmall-molecule-based therapeutic agents, nucleic acid-based therapeuticagents, viral vectors, and peptide based therapeutic agents. In oneexemplary embodiment, the therapeutic agent is a peptide basedtherapeutic agent. The terms “peptide,” “polypeptide” and “protein” areused interchangeably herein. Unless otherwise noted, the terms refer toa polymer having at least two amino acids linked through peptide bounds.The terms thus include oligopeptides, protein fragments, analogs,derivatives, glycosylated derivatives, pegylated derivatives, fusionproteins and the like. In another exemplary embodiment, the therapeuticagent is a corticosteroid pro-drug NSAID pro-drug, a ruboxistaurinpro-drug of the present invention, or a combination thereof. In certainexemplary embodiments, therapeutic loaded inner or outer particles arelyophilized.

Inner and outer particles are admixed together and exposed to asupercritical fluid under high pressure. In certain exemplaryembodiments, the supercritical fluid is carbon dioxide. Upon exposure tothe supercritical fluid the outer particles expand to create a porousstructure on the outer surface. The supercritical fluid then infuses theinner particles into the outer particles to form particle-in-particleextended release formulations. In one exemplary embodiment, theparticle-in-particle extended release formulations comprise theincorporation of inner nanoparticles having a diameter of approximately1 nm to approximately 900 nm in an outer microparticle having a diameterof approximately 1 μm to approximately 100 μm. In another exemplaryembodiment, the particle-in-particle extended release formulationscomprise the incorporation of an inner nanoparticle having a diameter ofapproximately 1 nm to approximately 300 nm in an outer nanoparticlehaving a diameter of approximately 10 nm to approximately 999 nm. In yetanother exemplary embodiment, the particle-in-particle extended releaseformulations include the incorporation of an inner microparticle havinga diameter of approximately 1 μm to approximately 100 μm in an outermicroparticle having a diameter of approximately 2 μm to approximately500 μm. Selection of an appropriate sized inner and outer particle willdepend on the type of material comprising the particles, the expansiveability of the outer partical in the supercritical fluid used, and thesize of inner particles to be incorporated within the outer particle.These are all factors that can be readily selected for by one ofordinary skill in the art. In general, the size ratio between the innerand outer particle may vary from approximately 1:2 to approximately1:100. In one exemplary embodiment the size ratio may be 1:5, 1:10,1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70,1:75, 1:80, 1:85, 1:90, 1:95, or 1:100

Formation of NPinPMPs may be achieved by exposure of the nanoparticlesand microparticles at approximately psi to approximately 1000 psi toapproximately 1400 psi. The time of exposure may vary from approximately5 minutes to approximately 2 hours. The temperature may range from 30°C. to 45° C. The selection of an appropriate pressure and temperaturerange are determined primarily by the range of temperature and pressuresnear the supercritical point for a given supercritical fluid.Accordingly, one of ordinary skill in the art will be able to select theappropriate time, temperature, and pressure ranged based upon thesupercritical fluid used, the size or amount of outer particle expansiondesired, and the degree of porosity in the outer particle desired. Forexample, exposure for longer periods of time and/or at higher pressuresfollowed by pressure quench will result in greater expansion andporosity than shorter exposure times and/or pressures.

In one exemplary embodiment, the inner particles and outer particles aremixed at a ratio of approximately 1:3. In one exemplary embodiment, theratio of inner particles to outer particles used is approximately 1:9.These ratios will influence the extent of nanoparticle incorporation andslow release of the drug. In general, the larger the amount of innerparticles relative to outer particles the higher the amount of innerparticles incorporated in outer particle, increasing the drug releaserates and the dose. The smaller the amount of inner particles relativeto the outer particles, the smaller the burst release.

The compositions and methods are further illustrated by the followingnon-limiting examples, which are not to be construed in any way asimposing limitations upon the scope thereof. On the contrary, it is tobe clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the spirit of the present invention.

EXAMPLES Example 1.1 Materials

Budesonide was purchased from Spectrum Chemical and Laboratory Products,a division of Spectrum Chemical Mfg. Corp. (New Brunswick, N.J., USA).Benzene, pyridine, triethylamine, sulfatrioxide triethylamine complex,and succinic anhydride were purchased from Sigma-Aldrich (St. Louis.Mo., USA). High performance liquid chromatography (HPLC) gradeacetonitrile and methanol were purchased from Fisher Scientific(Philadelphia, Pa., USA). Freshly excised bovine eyes were purchasedfrom G & C Meat Company, Colorado Springs, Colo., USA.

Synthesis of Budesonide Sulfate

To a solution of budesonide (50 mg; 0.116 mmoles) in 1 ml of anhydrousbenzene and pyridine (1:1), was added sulfatrioxide triethylaminecomplex (STT; 53.2 mg; 0.29 mmoles) in portions with stirring at 55-60°C. for 60 min (FIG. 1A). The formation of product was monitored every 10min with the help of TLC using chloroform-methanol (80-20) as mobilephase. After 1 h, the reaction mixture was evaporated for 2 h underreduced pressure to remove the solvent (benzene, B.P. 80° C.; pyridine,B.P. 116° C.) and a mixture of budesonide, budesonide-21-sulfatetriethylammonium salt and STT was obtained as oily residue. This mixturewas subjected to silica-gel open-column chromatography and furtherpurified by preparative thin layer chromatogarphy. Chloroform:methanolwas used as the mobile phase for product separation by chromatography.Product formation was confirmed by LC-MS/MS and NMR analysis.

Synthesis of Budesonide Succinate

100 mg of budesonide (0.2322 mmoles) was dissolved in 1 ml of anhydrouspyridine. 116 mg of succinic anhydride (1.16 mmoles) was added to theabove solution (FIG. 1B). The reaction mixture was stirred at roomtemperature for 18 h. There was slight formation of product as indicatedby TLC. The reaction temperature was then raised to 50° C. and allowedto stir for another 6 h. TLC and LC-MS/MS showed the formation ofproduct. The reaction mixture was added dropwise in 2 g ice+2 ml ofwater+1 ml of HCl in a beaker placed in an ice bath. The precipitatedproduct was collected by suction filtration. The solid was keptovernight for drying under vacuum in a dessicator. Product formation wasconfirmed by LC-MS/MS and NMR analysis.

Solubility Determination of Pro-drugs

Solubility of budesonide succinate was determined in phosphate bufferedsaline (PBS; pH 7.4) at 25° C. after adding the pro-drug at aconcentration of 1 mg/ml. At the end of 24 h, the suspension wascentrifuged at 15,000 rpm for 15 minutes at 4° C. using accuSpin Micro17(Fisher Scientific, USA). The pro-drug concentration in the supernatantafter filtration through 0.2 μm filter was determined using LC-MS/MS.

Solubility of budesonide succinate was determined in phosphate bufferedsaline (PBS; pH 7.4) at 25° C. after adding the pro-drug at aconcentration of 1 mg/ml. At the end of 24 h, the suspension wascentrifuged at 15,000 rpm for 15 minutes at 4° C. using accuSpin Micro17(Fisher Scientific, USA). The pro-drug concentration in the supernatantafter filtration through 0.2 μm filter was determined using LC-MS/MS.

Tissue Isolation from the Bovine Eye

Freshly excised bovine eyes were used in all studies. For isolation ofsclera and choroid-RPE (CRPE) (28), the anterior segment of the eye wasremoved with a circumferential cut below the limbus. The eye was cutinto two halves along the geometric axis, a line joining the anteriorpole (corneal center) and the posterior pole (center of the scleralcurve), and the vitreous was removed. Neural retina was removed byexposing the eyecup to isotonic assay buffer at pH 7.4. Equatorialregion of the remaining sclera-choroid-RPE (SCRPE) was used as is forSCRPE transport studies.

In Vitro Transccleral Transport of Budesonide/Budesonide Pro-drugs

Bovine sclera and sclera-choroid-RPE transport study was conducted asdescribed previously (28-30). Isotonic assay buffer (pH 7.4) with thefollowing composition was used during the entire tissue isolationprocedure and transport study: NaCl (122 mM), NaHCO₃ (25 mM), MgSO₄ (1.2mM), K₂HPO₄ (0.4 mM), CaCl₂ (1.4 mM), HEPES (10 mM), and glucose (10mM). After mounting the tissues in modified Using chambers, donorsolution (1.5 ml of 0.05 mM drug/prodrug) was filled in chambers facingthe episcleral side and receiver chambers were filled with the assaybuffer (pH 7.4) for A to B and vice-versa for B to A transport studies.p-amino hippuric acid (0.5 mM) was added in the transport study ofbudesonide succinate from A to B. The transport study was conducted for6 h at 37° C. under 95% air and 5% CO₂ aeration. Two hundred microlitersof sample was collected from the receiver side at specific timeintervals and replenished with fresh buffer. The drug/pro-drug contentin the receiver and donor samples was analyzed using an LC-MS/MS method.

Pharmacokinetic Model Development

Pharmacokinetic model was developed for three sets of budesonidesuccinate transport studies: A to B (sclera to RPE, FIG. 3A), A to B+MCTinhibitor (sclera to RPE, FIG. 4A) and B to A (RPE to sclera, FIG. 5A).The model was developed in such a way that it accounted for transport ofpro-drug from the donor to the receiver side, transport of parent drugformed in the donor chamber to the receiver side over the period oftransport study, loss of pro-drug/drug into the tissues and any backwardtransport from the receiver to the donor side. The transport rateconstants for parent drug and pro-drug were calculated from thecumulative percentage transport values. Rate constant for conversion ofpro-drug to the parent drug was calculated from the percentage of thepro-drug and parent drug formed. Model was run with the dose amountsobserved in the donor and receiver sides after 6 h transport studies.Final best fit model was run by DAMPING-GAUSS/SIMPLEX fitting algorithm,Fehlberg RFK 45 numerical integration method and equal weights forweighting of prodrug as well as parent drug transport.

Results

The final yields of the purified products were ˜50% (FIG. 1A).Budesonide sulfate formation was checked by LC-MS/MS (−Q1: m/z=509) and¹HNMR (D₂O, downfield shift of two protons on C 21 by 0.5 ppm afterintroduction of sulfate group).

Similarly, budesonide succinate formation was also confirmed by LC-MS/MS(−Q1: m/z=429) and ¹HNMR (CDCl₃), chemical shift values (δ, ppm) were asfollows: 0.960 (s, 3H), 1.5 (s, 3H), 2.9 (t, 4H, succinate protons), 6.0(s, 1H), 6.31 (d, 1H), 7.1 (d, 1H).

Solubility of budesonide succinate was found to be 51±3 μg/ml inphosphate buffered saline (pH 7.4) at 25° C.

In vitro transscleral transport of budesonide sulfate (1%) andbudesonide succinate (1.5%) were not significantly different (FIGS.2A-C). However, budesonide succinate transport was significantly(p<0.05) higher than budesonide (0.2%) across bovine sclera-choroid-RPEin sclera to RPE direction. Based on the higher cumulative percentagetransport observed with budesonide succinate, we selected this pro-drugfor our further investigations. Although a significant difference wasnot observed, transport of this pro-drug was reduced to 1% in presenceof a monocarboxylic acid transporter inhibitor in sclera to RPEdirection. Another important observation was that the transport ofbudesonide succinate was significantly higher (1.5%) in sclera to RPEdirection compared to RPE to sclera direction (0.5%).

FIGS. 3, 4 and 5 compare the model predicted and observed dose amountsof budesonide succinate and budesonide in donor and receiver chambersduring the course of study. Results have been presented in 3 separatefigures from a simultaneous model run which included the observed doseamounts of pro-drug as well as parent drug from the transport studies inA to B, A to B+inhibitor and B to A direction. The best fit model after20 runs had AIC value, R² and %CV of −33.65, 0.999 and ≦25%respectively.

Example 1.2 Method

All animals were handled according to the ARVO statement for the use ofAnimals in Ophthalmic and Vision Research. A suspension of budesonideand budesonide succinate (BSA, single prodrug of budesonide) was usedfor the ex vivo studies. one mg/ml suspension was made in sterilizedphosphate buffer saline (pH 7.4) in presence of carboxymethyl cellulosesodium salt (low viscosity, 50-200 cP; Sigma-Aldrich, Cat# C5678) at aconcentration of 0.5% w/v as a suspending agent. Drug suspensions wereshaken before each posterior subconjunctival injection. Rats weredivided into two groups. Group 1—Budesonide: animals were euthanizedwith 350 μl intraperitoneal injection of sodium pentobarbital (250mg/Kg). Twenty-five microliters of budesonide suspension was injectedusing a 30 G needle in the posterior subconjunctival space of one eye ofeach animal and the other eye was not treated. At the end of 1 h, eyeswere enucleated and immediately frozen in dry ice and isopentane bath.All samples were stored at −80 ° C. until LCMS/MS analysis. Group2—Budesonide succinate (BSA): animals were euthanized as mentionedabove. Immediately after euthanasia, animals were administered with 25μl suspension of budesonide succinate (1 mg/ml) in one eye via posteriorsubconjunctival injection using a 30 G needle and the other eye was leftuntreated. At the end of 1 h, the eyes were enucleated and frozen in asimilar manner as above until analysis. Periocular tissue samples werealso collected from both the groups at the end of the study. Differentocular tissues including sclera, choroid-RPE, retina, and vitreous wereisolated and analyzed for drug levels.

Results

The ex vivo drug levels in sclera, CRPE, retina, and vitreous for thebudesonide (Group 1) were 2.4, 3.0, 1.0, and 0.3 μg tissue. The ex vivodrug levels in sclera, CRPE, retina, and vitreous for the budesonidesuccinate (Group 2) were 1.9, 0.6, 0.2, and 0.05 μg/g tissue. The exvivo drug levels in the above tissues for budesonide released frombudesonide succinate (Group 2) were 11.0, 7.2, 2.2, and 0.5 μg/g tissue.Overall, the budesonide delivery in sclera, choroid-RPE and retina was2-5 folds higher with the prodrug, budesonide succinate (Group 2) ascompared to the plain drug budesonide (Group 1). Results are shown inFIGS. 6 and 7

Example 2 Materials and Methods

Celecoxib was purchased from Spectrum chemical and laboratory products(New Brunswick, N.J., USA). Celecoxib succinamidic acid (C-SA, singlepro-drug of celecoxib) and celecoxib succinamidic acid taurine (C-SA-T,double pro-drug of celecoxib) were synthesized in the laboratory.Succinic anhydride, dimethylamino pyridine, triethylamine, carbonyldiimidazole, N,N dimethyl formamide (anhydrous), tetrahydrofuran(anhydrous), taurine, natural melanin (isolated from Sepia officinalis),triton-X and EDTA were purchased from Sigma Aldrich (St. Louis. Mo.,USA). All reagent grade organic solvents such as chloroform,dichloromethane, ethyl acetate, methanol and acetone were purchased fromACROS ORGANICS (New Jersey, USA). PBS pH 7.4 was used as a vehicle forall titrations and in vitro/in vivo studies. Freshly excised bovine eyeswere purchased from G & C meat company, Colorado Springs. MaleBrown-Norway (BN; pigmented) rats weighing 150 to 200 g were purchasedfrom Charles River Laboratories, Wilmington, Del., USA.

Synthesis of celecoxib-sussinamidic acid pro-drug (C-SA)

Celecoxib (2.62 mmoles) was dissolved in anhydrous THF under argonatmosphere. DMAP and TEA (3.15 mmoles each) were added while stirring.After 5-10 minutes, succinic anhydride (3.15 mmole) was added andreaction mixture was stirred overnight (16-18 h) at room temperatureafter which it was refluxed for 5-6 h (FIG. 8). At the end of 24 h, whenthere was no further increase in the intensity of the TLC spot for theproduct and the reaction was stopped. The organic layer was collectedand concentrated to a viscous material on rotary evaporator. The viscousmaterial was dissolved in minimum volume of dichloromethane and washedonce with equal volume of water. Final product in the organic layer waspurified by column chromatography. Silica gel 60 (Geduran, particle size40-63 μm, EMD Chemicals) and dichloromethane/methanol were used asstationary phase and mobile phase respectively.

Synthesis of Celecoxib-Succinamidic acid-Taurine Prodrug (C-SA-T)

This is a two-step process starting from C-SA prodrug as shown in FIG.8. Terminal carboxylic acid functionality in celecoxib-succinamidic acid(1 mmole) was first activated with carbonyl diimidazole (CDI) and thenreacted with taurine in presence of triethylamine as catalyst. One mmoleof C-SA was dissolved in anhydrous dimethyl formamide (DMF) and CDI (1.5mmoles) dissolved in anhydrous DMF was added dropwise at 0° C. over aperiod of 1 h. The reaction mixture was stirred for another 1 h at 0° C.Taurine (2 mmoles) suspended in minimum volume of water along withtriethylamine (2 mmoles) was added dropwise to the above reactionmixture and stirred at room temperature for 24 h. The reaction mixturewas concentrated under high vacuum. The solid mixture was dissolved indichloromethane-acetonitrile-water (90:5:5) and purified product wasobtained after column chromatography as done previously for CSA.

In Vitro Transport Across Bovine Cornea, Conjunctiva andSclera-Choroid-RPE

Freshly excised bovine eyes procured from a local slaughter house wereused for all in vitro transport studies. Fatty or adherent tissue allaround the eye is removed first. Cornea and conjunctiva were thenremoved. For isolation of sclera and choroid-RPE (CRPE), the anteriorsegment of the eye was removed with a circumferential cut below thelimbus. The eye was cut into two halves along the geometric axis, a linejoining the anterior pole (corneal center) and the posterior pole(center of the scleral curve), and the vitreous was removed. Neuralretina was removed by exposing the eyecup to isotonic assay buffer at pH7.4. Equatorial region of the remaining sclera-choroid-RPE (SCRPE) wasused as is for SCRPE transport studies. All transport studies forcelecoxib were performed in presence of 5% HP-β-CD. Transport of C-SA-Twas also performed in B to A direction across SCRPE. Drug solutions wereprepared in the following manner: Celecoxib (100 μg/ml) in presence of5% HP-β-CD was made after triturating celecoxib with HP-β-CD in apestle-mortar followed by addition of assay buffer. The mixture wasstirred for 24 h. Before use, the clear solution was filtered through0.2 μ filter. C-SA and C-SA-T (100 μg/ml) pro-drug solutions were madejust before the start of the experiment followed by filtration through0.2 μ filter. All transport experiments were performed for 6 h at 37° C.Samples were collected at regular time intervals from the receiver sidewith replacement with fresh assay buffer (pH 7.4). Transport studieswith all solutes were performed in A to B direction meaning outward toinward (eg. sclera towards choroid-RPE) movement of the solute wasmeasured. Transport for C-SA-T was also performed in B to A directionmeaning inward to outward movement (choroid-RPE towards sclera) for thissolute was also measured. Samples from all the transport studies wereanalyzed on LC-MS/MS.

Isothermal Titration Calorimetry for Melanin Binding Estimation

Following parameters were used for calculating the melanin binding ofcelecoxib/C-SA in the ITC titration: melanin=1 mg/ml suspension in PBS(pH 7.4), celecoxib=2 μg/ml in PBS (pH 7.4) and CSA=250 μg/ml in PBS (pH7.4), 60 injections of 5 μl each, injection duration=10.3 sec, spacingbetween two consecutive injections=300 sec, stirring speed=595 rpm,temperature=37° C. All samples were degassed before ITC injections. Forcalculating the heat of dilution, celecoxib/C-SA was titrated againstPBS using the same injection parameters.

In vivo Delivery Assessment of Celecoxib/C-SA/C-SA-T Eye Drops in MaleBrown Norway (BN) Rats

Two mg/ml suspension of celecoxib, 2 mg/ml solution of C-SA and C-SA-T,10 mg/ml solution of C-SA-T was made in PBS pH 7.4. 10 μl eye drop ofcelecoxib (n=6 eyes; 3 animals), C-SA and C-SA-T (n=8 eyes; 4 animals)was administered to both the eyes of each BN rat. At the end of 1 h, theanimals were euthanized and the eyes were enucleated. Periocular tissueand plasma samples were collected.

Sample processing: Acetonitrile precipitation method was used for drugextraction from the tissues. Briefly, eyes from each eye drop study weredissected for tissue isolation. Tissues were homogenized in 250 μl water(pH 7.4 adjusted with 5 mM ammonium acetate) containing indoprofen asinternal standard at a concentration of 500 ng/ml (1000 ng/ml stock wasmade). Similar volume of acetonitrile was added and mixed well byvortexing for 30 minutes. Samples were centrifuged at 15,000 rpm for 30minutes. Supernatant was collected for LC-MS/MS analysis.

LC-MS/MS Method

Mass parameters: The mass spectrometric parameters of celecoxib, C-SA,C-SA-T and indoprofen (internal standard) were optimized in negativeionization mode by infusing a 1.0-μg/mL solution on a liquidchromatography tandem mass spectrometry instrument (API 3000; PE SCIEX,Concord, Ontario, Canada) by the syringe infusion mode. All analyteswere monitored in multiple reaction monitoring (MRM) mode (celecoxib380/316, C-SA 480/380, C-SA-T 587/206 and indoprofen 280/236). LCparameters: Zorbax SB C18 (3.5 μm, 2.1×100 mm) column; 5 mM ammoniumformate (pH 6.8, mobile phase A) and acetonitrile (mobile phase B) asmobile phase in gradient elution mode; flow rate 0.4 ml/min, columntemperature 25° Celsius. Total run time was 5.5 min.

Effcacy Assessment of C-SA and C-SA-T Eye Drops inStreptozotocin-Induced Diabetic Retinopathy Rat Model

Diabetes Induction: BN rats weighing 175-225 g were acclimatized for atleast two days before any experimental procedure. After overnightfasting for 12-16 h, an intraperitoneal injection of 30 mg/ml solutionof streptozotocin in 10 mM citrate buffer (pH 4.5) was administered (60mg/kg body weight) to induce diabetes. After 3-4 h of streptozotocininjection, animals were put on regular diet and 24 h afterstreptozotocin injection, blood sample (5-10 μl) was collected via tailvein. The blood glucose levels in the animals were determined with aglucose monitor (One Touch; Life Scan Inc., Milpitas, Calif.). Animalswith blood glucose levels greater than 250 mg/dL were considereddiabetic. The animals were divided into four groups. Group 1: Normal(N=12), Group 2: Diabetic+vehicle (N=12), Group 3: Diabetic+0.2% w/vC-SA eye drops (N=12) and Group 4: Diabetic+1.0% w/v C-SA-T eye drops(N=12). Treatment was started immediately after diabetes induction. Botheyes were dosed twice daily for 60 days in group 2, 3 and 4 with theirrespective treatment. Animals in groups 2-4 were sacrificed at the endof 2 months. Normal animals in group 1 were sacrificed immediately afteracclimatization in the animal facility. For each of the three assays,namely, FITC-Dextran leakage, vitreous-to-plasma protein ratio, andleukostasis, we used 4 animals from each of the four groups mentionedabove.

Blood Retinal Barrier Leakage

Retinal FITC-Dextran Leakage: BN rats from each group were used for theassessment of FITC-dextran leakage. At 1 hour after last dosing on day61, rats were sacrificed for FITC-dextran leakage assay. Brief protocolfor the assay and tissue sample processing is described below. First,the animals were anesthetized with ketamine (80 mg/kg) and xylazine (12mg/kg) administered intraperitoneally. Then a 50 mg/ml PBS (pH 7.4)solution of FITC-dextran with a molecular weight of 4.4 kDa wasadministered (50 mg/kg body weight) intravenously via tail vein. Animalswere euthanized with 150 mg/kg sodium pentobarbital after 10 minutes(circulation time for FITC-dextran) of tail vein injection. Bloodsamples (0.5-1 ml) were withdrawn from the heart in 2 ml Eppendorf tubes(SureLock Microcentrifuge Tubes, LIGHTLABS, USA) containing 50 μl ofEDTA. Chest cavity was opened. Animals were perfused with PBS (500 ml/kgbody weight) for 6-7 minutes after a 20G needle attached to a 50 mlsyringe was inserted into the left ventricle. Eyes were enucleated andisopentane-dry ice bath was used to immediately snap-freeze the eyesbefore storing them at −80° C. Retina of each eye was isolated, weighedand homogenized in 500 μl of PBS (pH 7.4). Following homogenization, 500μl of PBS containing 2% Triton X-100 was added to the homogenate. Themixture was vortexed at room temperature for 1 h. The homogenate wascentrifuged at 15000 rpm (21,130 g) for 20 min and the supernatant wascollected. The relative FITC-dextran fluorescence units in 1 ml ofsupernatant were measured using a spectrofluorometer set at anexcitation wavelength of 483 nm and an emission wavelength of 538 nm.Fluorescence of blank PBS was also

$\frac{{{Retinal}\mspace{14mu} {FITC}} - {{dextran}\mspace{14mu} {({\mu g})/{retinal}}{\mspace{11mu} \;}{weight}\mspace{14mu} (g)}}{{{Plasma}\mspace{14mu} {FITC}} - {{{dextran}{\mspace{11mu} \;}\left( {{\mu g}/{\mu l}} \right)} \times {ci}\#}}$

measured for subtraction from each sample reading. Final concentrationof FITC-dextran was expressed as μg/g tissue. Standard curve wasgenerated using known amounts of FITC-dextran (5 ng/ml to 5 μg/ml). 20 μ(˜20 mg) of plasma was diluted to 1 ml (50 times dilution) with PBS forquantification under the linear range of the standard samples. Thedilution factor was taken into account for estimating the amount ofFITC-dextran per microliter of blood sample. The amount of FITC-dextranleakage in to the ocular tissues was calculated using the followingequation, after correcting for dilutions.

Vitreous-to-Plasma Protein Ratio: BN rats from each group were used forthe assessment of vitreous to plasma protein ratio. At 1 h after lastdosing on day 61, rats were sacrificed for determining the proteinratio. Rats were euthanized with 150 mg/kg sodium pentobarbitaladministered intraperitoneally. Eyes were enucleated and isopentane-dryice bath was used to immediately snap-freeze the eyes before storingthem at −80° C. Blood samples (0.5-1 ml) were withdrawn from the heartfollowing cardiac puncture in 2 ml Eppendorf tubes (SureLockMicrocentrifuge Tubes, LIGHTLABS, USA) containing 50 μl of EDTA. Theabove samples were centrifuged at 15,000 g at 4° C. for 15 min tocollect the plasma in the supernatant. Plasma samples were stored at−80° C. Ocular tissues including the retina and the vitreous from eacheye were isolated and weighed. The vitreous was allowed to liquefy. Thevitreous samples were centrifuged at 15,000 g at 4° C. for 20 min. Thesupernatant of vitreous was collected (20 μl) in new Eppendorf tubes andweighed (weight range=19-21 mg). The supernatant (20 μl) was diluted to1 ml with PBS (pH 7.4) (50 times dilution). One hundred microliters ofthe above diluted material was mixed with 1 ml of Bradford reagent.Absorbance of the above 1 ml volume was measured at 595 nm. The plasmasample (20 μl) was also diluted to 1 ml with PBS (pH 7.4) (50 timesdilution). One hundred μl of the above diluted material was mixed with 1ml of Bradford reagent. Absorbance of the above 1 ml volume was measuredat 595 nm. The standard curve was generated using known concentrationsof bovine serum albumin (25-500 μg/ml in PBS, pH 7.4). Hundredmicroliters of each standard was mixed with 1 ml of Bradford reagent.The amount of protein in plasma and vitreous was estimated from thestandard curve after correcting for dilutions.

Retinal Leukostasis

BN rats from each group were used for the assessment of adherentleukocytes. At 1 hour after last dose on day 61, rats were sacrificedfor ex-vivo retinal leukostasis assay. First, the animals wereanesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg)administered intraperitoneally. Then, the chest cavity was carefullyopened and animals were perfused with PBS (250 ml/kg body weight) for6-7 minutes after inserting a 20G needle attached to 50 ml syringe intothe left ventricle. Animals were then perfused with a 40 μg/ml PBS (pH7.4) solution of FITC-conjugated concanavalin A lectin (5 mg/kg, ˜33 ml)to label the adherent leukocytes and the vascular endothelial cells.Animals were perfused again with similar volume of PBS as above toremove unbound lectin. Eyes were enucleated and fixed in 2%paraformaldehyde for 2 h. Retinas were carefully removed to prepare theflat mounts. Fluorescence microscope (Digital Eclipse Cl; Nikon Inc.,Melville, N.Y.) under blue light (Ex 465-495, DM 505, BA 515-555) with a20× objective was used to count the number of leukocytes adhered to thevessel walls. The count was compared between treated and untreated rats.

Statistical Analyses

All data in this study are expressed as the mean±S.D. Comparisonsbetween two groups were done by Student's t-test whereas comparisonsamong multiple groups were done using one-way ANOVA followed by a Tukeypost hoc analysis. Statistical significance was set at p≦0.05

Results

Synthesis and characterization of celecoxib-succinamidi acid (C-SA) andCelecoxib-Succinamidic Acid-Taurine (C-SA-T) Pro-Drugs.

The overall yield of C-SA (single amide prodrug of celecoxib) was75-80%. Structure of C-SA was confirmed from the spectral data of ¹HNMRand MS (TOF, ES). ¹HNMR (CDCl₃) δ 2.3 (s, 3H), 2.5 (t, 2H), 2.7 (t, 2H),6.8 (s, 1H), 7.0 (d, 2H), 7.2 (d, 2H), 7.4 (d, 2H), 7.8 (d, 2H), 9.9 (s,1H). MS (TOF, ES) (m/z) 479.97, 379.99. The overall yield of C-SA-T(double amide prodrug of celecoxib) was 55-60%. Structure of C-SA-T wasconfirmed from the spectral data of ¹HNMR and MS (TOF, ES). ¹HNMR(CDCl₃) δ 2.3 (s, 3H), 2.5 (t, 2H), 2.7 (t, 2H), 3.6 (t, 2H), 3.7 (t,2H), 6.8 (s, 1H), 7.0 (d, 2H), 7.2 (d, 2H), 7.4 (d, 2H), 7.8 (d, 2H),9.9 (s, 2H) MS (TOF, ES) (m/z) 587.04, 480.01, 380.04.

In vitro Transport Across Bovine Cornea, Conjunctiva and Sclera-ChoroidRPE

Cumulative percent transport (FIG. 9) of celecoxib (5% HP-β-CD), C-SAand C-SA-T at the end of 6 h across bovine cornea was 0.017, 0.177 and0.441% respectively. Transport of C-SA and C-SA-T was found to beapproximately 10- and 25-times higher than celecoxib. Transport ofcelecoxib (5% HP-β-CD), C-SA and C-SA-T across bovine conjunctiva was3.9, 8.4 and 15.6% respectively. Transport of C-SA and C-SA-T was foundto be approximately 2- and 4-times higher than celecoxib. Transport ofcelecoxib (5% HP-βCD), C-SA and C-SA-T across bovine sclera-choroid-RPEwas approximately 0.1, 1.0, and 2.0% respectively. Transport of C-SA andC-SA-T was found to be approximately 10- and 20-times higher thancelecoxib. The effect of taurine as an inhibitor was seen with a>50%reduction in the transport of C-SA-T across cornea and SCRPE.

Melanin Binding Estimation Using Isothermal Titration Calorimetry

FIGS. 10A-C represents the melanin binding of celecoxib, C-SA and C-SA-Twith natural melanin. The value of binding/association constant forcelecoxib with melanin was found to be Ka=1.01×10⁶M⁻¹. Whereas, similarvalue for C-SA and C-SA-T binding with melanin was found to be8.89×10³M⁻¹ and 7.39×10³ M⁻¹ respectively, which was approximately 110to 130 fold lower than melanin binding affinity of celecoxib.Furthermore, we used C-SA at a concentration 125 times higher thancelecoxib for our binding experiments. In generating the pro-drug , aslightly basic and positively charged sulphonamide group of celecoxibwas converted into acidic/negatively charged succinamidic acid. It isgenerally believed that acidic/negatively charged groups bind less withmelanin as compared to basic/positively charged groups.

In vivo Delivery of Celcoxib/CSA as Eye Drops in Brown Norwary Rats

Levels of celecoxib in cornea was higher than CSA which may beattributed to the highly lipophilic nature of celecoxib. Anotherimportant factor may be the viscosity of celecoxib suspension which wasobserved to be much higher than that of CSA. Similarly, levels ofcelecoxib were higher in iris and CRPE than C-SA possibly because of thehighly pigmented nature of these tissues. However, levels of celecoxibin periocular space were observed to be lower than that of CSA. In allother tissues except for cornea, iris and CRPE, levels of CSA werehigher than that of celecoxib. Retinal delivery was almost double withC-SA when compared to celecoxib. Levels of celecoxib with C-SA-Tpro-drug were higher in all tissues when compared with the parent drug.Enhancement in retinal delivery with 0.2% w/v and 1% w/v C-SA-T aftertopical administration was found to be about 10 and 20 foldsrespectively compared to celecoxib.

Efficacy Assessment of C-SA and C-SA-T Eye Drops inStreptozotocin-Induced Diabetic Retinopathy Rat Model.

Blood retinal barrier leakage was assessed via retinal FITC-dextranleakage and vitreous-to-plasma protein ratio. As represented in FIG.13A, diabetic animals treated with vehicle (PBS, pH 7.4) had almost4-fold higher retinal barrier leakage (mean± s.d.=97.80±34.59 μl/g/min)at the end of 2-months when compared to normal animals (mean±s.d.=22.83±7.21 μl/g/min). Animals treated with 0.2% w/v C-SA solutiondid not show any significant reduction in the leakage (mean±s.d.=93.66±37.08 μl/g/min). On the other hand, animals treated with thedouble pro-drug of celecoxib (C-SA-T, 1.0% w/v solution) showedsignificant reduction in the leakage (mean± s.d.=23.64±4.20 μl/g/min).C-SA-T was able to bring down the leakage levels close to that assessedin the normal animals.

Vitreous-to-Plasma Protein Ratio

As represented in FIG. 13B, normal animals demonstrated an averagevitreous-to-plasma protein ratio of 0.14±0.03, whereas diabetic animalshad an average value of 0.54±0.10, approximately 4-fold higher thannormal animals. Animals treated with 0.2% w/v C-SA solution demonstratedan average vitreous-to-plasma protein ratio of 0.49±0.11, which was notsignificantly different than diabetic animals. On the other hand,animals treated with 1.0% w/v C-SA-T solution demonstrated an averagevitreous-to-plasma protein ratio of 0.28±0.06 which was significantlylower than the diabetic animals treated with plain vehicle. The rankorder for the vitreous-to-plasma protein ratio among various groups ofanimals was: diabetic≧diabetic+0.2% w/v C-SA treatment>diabetic+1.0% w/vC-SA-T treatment≧normal (Student's t-test, p-value<0.001).

Retinal Leukostasis

Adhesion of leukocytes to the retinal vasculature is another marker ofthe disease progression. Leukocyte count was monitored underfluorescence microscope for all groups of animals. Mean retinalleukocyte count in normal, diabetic+vehicle, diabetic+0.2% w/v C-SA, anddiabetic+1.0% w/v C-SA-T groups was found to be 32±7, 114±19, 95±12, and47±10, respectively, which clearly indicated that the double prodrug ofcelecoxib (C-SA-T) was effective in reducing the retinal leukocytes indiabetic retinopathy. The rank order for the retinal leukostasis amongvarious groups of animals was: diabetic≧diabetic+0.2% w/v C-SAtreatment>diabetic+1.0% w/v C-SA-T treatment≧normal (Student's t-test,p-value<0.001). See FIG. 13C.

Example 3

Plain polymeric PLGA microspheres and PLA nanospheres were preparedusing an emulsion solvent evaporation method. Plain PLGA microsphereswere prepared by dissolving 100 mg of polymer in 1 ml of DCM followed bydispersion of polymer solution into 10 ml of 2% aqueous poly vinylalcohol solution under homogenization at 10,000 rpm for 1 min(Virtishear Cyclone®, USA). The prepared O/W emulsion was furthertransferred into 100 ml of 2% aqueous poly vinyl alcohol solution underhomogenization at 15000 rpm for 5 min using a Virtishear Cyclone®Homogenizer. The organic solvent was then evaporated from the final 2%aqueous polyvinyl alcohol solution by stirring for 3 hr at roomtemperature. Subsequently, the microspheres were separated bycentrifugation (Beckman, USA) at 12000 rpm for 15 min at 4° C. Themicrosphere pellet was suspended in 50 ml of distilled water andlyophilized (Lanconco Triad, USA) for 24 hr to obtain dry microspheres.Plain PLA nanospheres were prepared by dissolving 100 mg of polymer in 1ml of DCM and 200 μl of water was dispersed into polymer solution andsonicated (Misonix Inc., USA) for 1 min at a power level of 10 W on iceto prepare the primary emulsion. This was later dispersed in 50 ml of 2%aqueous polyvinyl alcohol and sonicated for 5 min at 30 W. Subsequently,the PLA nanospheres were collected, and lyophilized by a similarprocedure described above.

Example 4

Supercritical fluid pressure quench technology was used for expansionand porosification of blank PLGA microspheres and then bevacizumab wasfilled into porous microspheres. Briefly, 50 mg of plain PLGAmicrospheres were placed in a high pressure vessel and exposed to SC CO₂at a pressure of 1150-1200 psi and a temperature of 33° C. for 30 min.After completion of the SC CO₂ exposure, the pressure was released overa minute and the particles were collected. Subsequently, bevacizumab wasfilled into pores by incubating 100 μl of bevacizumab solutionequivalent to 2.5 mg for 30 min and lyophilized overnight. In vitrorelease of bevacizumab was carried out in PBS pH 7.4 and bevacizumabcontent was estimated using micro BCA assay (FIG. 14).

Example 5

In an another approach, bevacizumab was encapsulated into PLGAmicrospheres by first coating 2.5 mg of bevacizumab in 100 μl on 50 mgof plain PLGA microspheres through lyophilization followed by theirexposure to supercritical CO₂ as explained example 4. In vitro releaseof bevacizumab was carried out in PBS pH 7.4 and bevacizumab content wasestimated using micro BCA assay (FIG. 15).

Example 6

A novel supercritical infusion and pressure quench technology wasdeveloped for preparing nanospheres in porous microspheres (NPinPMP) inorder to sustain bevacizumab release. In this technology, plain PLAnanospheres were coated with bevacizumab by lyophilization and furthermixed with plain PLGA microspheres and exposed to supercritical CO_(2.)Briefly, 100 μl of bevacizumab solution (2.5 mg) was added to 50 mg ofPLA nanospheres and incubated at 4° C. for 30 min and lyophilizedovernight (B-PLA NP). Later, bevacizumab coated PLA nanospheres weremixed with plain PLGA microspheres and placed in a high pressure vessel.The particles were exposed to SC CO₂ at a pressure of 1150-1200 psi anda temperature of 33° C. for 30 min. After SC CO₂ exposure, the pressurewas released over a minute and the particles were collected. In vitrorelease of bevacizumab was carried out in PBS pH 7.4 and bevacizumabcontent was estimated using micro BCA assay (FIG. 16). The bevacizumabactivity was measured by ELISA method. The bevacizumab conformationaland structural stability in the in vitro release study was evaluated bysize exclusion chromatography, circular dichorosim spectroscopy, and bygel electrophoresis. The in vivo delivery of bevacizumab was monitorednon-invasivley after intravitreal injection in rat model.

Activity of Bevacizumab in Release Samples

Activity of bevacizumab released from NPinPMP was evaluated by sandwichELISA method. The ELISA plate (BD life sciences, USA) was coated with100 μl of 0.1 μg/ml of VEGF-165 in 50 mM sodium carbonate buffer, pH 9.6and incubated overnight at 4° C. After overnight incubation, the platewas washed with wash buffer thrice, blotted, and air dried. Afterwards,300 μl of blocking solution (0.5% BSA & 0.05% Tween 20 in PBS pH 7.4)was added to each well and incubated in dark for 1 hr. Then, the platewas washed with wash buffer thrice, blotted, and dried. The bevacizumabstandards were prepared (0.5-50 ng/ml) in dilution buffer and incubatedin dark for 2 hr. Subsequently, 100 μl of each released sample was addedto the respective wells. After 2 hr incubation, plate was washed withwash buffer thrice, blotted, and dried. The secondary goat anti-humanIgG (FC) antibody was diluted (1:10000) in TBS (Tris-buffered saline) pH7.6-7.8 with 1% BSA and 100 μl of this solution was added to the plateand incubated in dark for 2 hr. After incubation, the plate was washedthrice with wash buffer and dried. To each well 100 μl of TMB substrate(3,3′,5,5″-tetramethylbenzidine) was added and left for colordevelopment. After 30 min incubation, 50 μl of stop solution was addedand absorbance was recorded at 450 nm. A similarly processed standardcurve was used to quantify bevacizumab in the released samples. As shownin FIG. 19, released bevacizumab is active and has retained affinity forVEGF-165 confirming that the released bevacizumab retained bindingability to VEGF-165 for 4 months.

Stability Evaluation of Bevacizumab in Release Samples by Size ExclusionChromatography (SEC)

Formation of soluble aggregate sand degradation products of bevacizumabreleased from NPinPMP was evaluated using size exclusion chromatography(SEC). A silica based size exclusion column (TSK® Gel G3000SWX) having 5μm particle diameter, with dimensions of 7.8 mm×30 cm, and pore size of250 Angstroms was used. The mobile phase was an aqueous solution of0.182 M KH2PO4, 0.018 M K2HPO4 and 0.25 M KC1 at pH 6.2. Flow rate ofthe mobile phase was 0.50 ml per minute. A UV detector scanning over thewavelength of 210-400 nm was used to detect the eluents from the sizeexclusion column.

As showin in FIG. 21, analyses of SEC chromatograms of releasedbevacizumab showed the presence of a single prominent monomeric peak ofbevacizumab at 8.1 min. The single observed peak suggests that physicaland chemical stability of bevacizumab was maintained at 37° C. over a 4month release time.

Conformational Stability Evaluation of Bevacizumab in Release Samples byCircular Dichroism (CD)

The change in the secondary structure of bevacizumab after SC CO₂exposure and in the in vitro release samples was determined usingcircular dichorism (CD) spectroscopy (Photophysics, USA). The samples atequal protein concentrations were taken in a stain free quartz cuvettewith a path length of 1 mm and spectra were recorded at 25°‘C. The datawas collected at 1 nm step size in the 200-260 nm wavelength region.

As shown in FIG. 20, Circular dichroism spectrum of native bevacizumabshow a peak at 218 nm, indicating the presence of a higher percentage ofbeta sheet like structures, which is consistent with the rich beta-sheetrich conformations of known antibodies. Released bevacizumab fromexemplary PinPs at 1, 2, 3 and 4 month time points showed CD spectrasimilar to native bevacizumab. The observed spectra indicate that thebevacizumab's secondary structure was not significantly changed duringthe release study.

Evaluation of Bevacizumab Degradation in Release Samples by GelElectrophoresis

The degradation and aggregation of bevacizumab from in vitro releasesamples was assessed by both reducing and non-reducing gelelectrophoresis. Gel electrophoresis was performed using 4-20% SDS-PAGEprecast gradient gel. Samples were prepared by taking 30 μl of eachsample equivalent to 10 μg of bevacizumab and 15 μl of 2× loading dyefollowed by boiling for 5 min and then centrifuged at 15000 rpm for 5min (Beckman Avanti 30, Beckman Coulter, Inc. USA). Each sample (40 μl)was loaded on precast gel and electrophoresed for 2 hr at 20 mA.Subsequently, the gel was stained with Coomassie Blue R-250, de-stainedand visualized under Gel-DOC system (Bio-Rad Laboratories, USA).

As shown in FIGS. 23A-B, the SDS-PAGE data indicate the absence ofdegradation and/or aggregation of bevacizumab in the release samplesover a 4 month period.

In vivo Delivery of Bevacizumab in Rats

In vivo delivery of bevacizumab was evaluated following intravitrealadministration of Alexa Fluor 488 conjugated bevacizumab in NPinPMP in arat model. Rats were anesthetized by intraperitoneal injection ofketamine (35 mg/kg)/xylazine (5 mg/kg) and once the rats were underanesthesia, betadine solution was applied on the eye surface, andintravitreal injections were made using a 30-G needle. The rat eyes wereinjected with Alexa- bevacizumab encapsulated NPinPMP formulation (1.8μg of Alexa-bevacizumab plus unlableled bevacizumab of 5.4 μg in/5 μl;300 mg particles/1 ml PBS pH 7.4) and as a control Alexa-bevacizumab atequivalent concentration (7.2 μg/5 μl) was injected. Ocular fluorescencedue to the release of Alexa-bevacizumab was monitored periodically usingFluorotron Master™ (Ocumetrics, CA, USA) until the fluorescence reachedthe lower detection limit or baseline. Baseline fluorescence values ofeyes were monitored before injecting the formulations. At each timepoint, three fluorometric scans were taken and the mean value was used.Standard curve for Alexa-bevacizumab at different concentrations wasobtained using a cuvette and ocular flurophotometry with a rat lensadapter. The standard curve was used to convert fluorescein equivalentconcentrations provided by fluorophotometer to actual Alexa-bevacizumabconcentration.

After intravitreal injection of Alexa-bevacizumab encapsulating NPinPMP,and soluble alexa-bevacizumab, the concentration distribution ofbevacizumab along the eye optical axis was determined indirectly bymeasuring the alexa fluorescence intensity distribution (equivalent ofsodium fluoresciene concentration) curve along axial planes, indicatedas data points in an anterior to posterior direction. The fluorescencescans revealed sustained delivery of Alexa-bevacizumab from NPinPMPcompared to solution. Fluorescein equivalent concentrations reported byFluorotron Master were converted to Alexa-bevacizumab concentrations.The Alexa-bevacizumab concentration in the vitreous region from solutionand NPinPMP group at different time points was plotted. Only theconcentrations of the labeled bevacizumab are reported.

Before intravitreal injection, the baseline fluorescence readings ofnormal eyes were taken and the baseline fluorescence concentration wasfound to be 1.78 μg/ml. As shown in FIGS. 24A-C, the Alexa-bevacizumabsolution injected group showed Alexa-bevacizumab concentration of 32μg/ml on day 1 and reduced to 2.2 μg/ml by day 15 indicating rapidelimination from vitreous region. In NPinPMP injected group theAlexa-bevacizumab concentration in the vitreous on day 1 was found to be5 μg/ml and the Alexa-bevacizumab concentration was 3.5 μg/ml on day 45.However, by the end of day 60, the Alexa-bevacizumab concentration wasobserved to be 2.2 μg/ml, reaching baseline reading. The observed dataindicate an ability to achieve sustained in vivo delivery of bevacizumabfrom an exemplary PinP composition.

Example 7

Confocal microscopy study was used to confirm the infusion ofbevacizumab coated PLA nanospheres inside the porous PLGA microspheresby SC CO₂ treatment. Nile red loaded PLA nanospheres and 6-coumarinloaded PLGA microspheres were prepared using emulsion solventevaporation method. Nile red and 6-coumarin (100 μg/100 mg polymer) weredissolved in polymer solution of PLA and PLGA, respectively before theparticle preparation. 6-coumarin loaded PLGA microspheres and themixture of Nile red loaded PLA NP and 6-coumarin loaded PLGA MP at aweight ratio of 1:9 were subjected to SC CO_(2.) The SCF conditions wereabout 1200 psi for 30 minutes at 33° C. The SC CO₂ treated particleswere observed under confocal microscopy (Leica Microsystems, USA) atdifferent magnifications (10, 20 & 100×). Further, Z-stack confocalimaging was adopted to capture the localization of Nile red in the6-coumarin loaded PLGA microspheres. Images were captured at an intervalof 0.25 μm. Nile red excitation was done at 561 nm and fluorescenceimages were captured using a red filter. Similarly, 6-coumarinexcitation was done at 488 nm and the fluorescence image was capturedusing a green filter.

The confocal images of expanded porous PLGA MP and NPinPMP formulationwere shown in FIGS. 17 and 18 and indicated the expansion of PLGAmicrosphere with pore formation after SC CO₂ exposure. After SC CO₂treatment, the red signal of Nile red was observed to be surrounded bygreen signal of 6-Coumarin indicating the infusion of PLA NP inside theexpanded PLGA MP. The Z sectioning image of NPinPMP formulation showedthe localization of Nile red loaded NP inside the expanded 6-Coumarinloaded PLGA microspheres.

Surface morphology of gold coated PinP, were visualized using a scanningelectron microscope (JSM-6510, Jeol USA, Inc., CA) at differentmagnifications ranging from 1000 × to 5000 ×. As shown in FIG. 19, SCFleads to the expansion of PLGA MP with pore formation. Also, from theSEM images it was evident that PLA NPs were encapsulated inside theexpanded porous PLGA MP.

Example 8

The applicability of PinP for protein sustained release was evalautedusing another protein His-LEDGF₁₋₃₂₆. NPinPMP encapsulatingHis-LEDGF₁₋₃₂₆ were prepared as explained in Example 6. In vitro releaseand in vivo delivery of His-LEDGF₁₋₃₂₆ from NPinPMP was performed.

In vitro Cumulative Release of His-LEDGF₁₋₃₂₆

His-LEDGF₁₋₃₂₆ encapsulated NPinPMP were evaluated for in vitro releasein PBS pH 7.4. Particles (2-3 mg) were weighed and dispersed in 1 ml ofPBS pH 7.4 and incubated at 37° C. under shaking at 200 rpm (Max Qshaker incubator). At pre-determined time points the suspended particleswere centrifuged at 13,000 g for 15 min and the supernatant wascollected. The pellet comprising particles was re-suspended in 1 ml offresh PBS pH 7.4 and incubated. The His-LEDGF ₁₋₃₂₆ content in thesamples was estimated using micro BCA assay as per the manufacturer'sinstructions (Pierce Biotechnology, IL, USA). The in vitro cumulativedata showed the sustained release of His-LEDGF₁₋₃₂₆ from NPinPMP. Asshown in FIG. 25, a cumulative 60% release of His-LEDGF₁₋₃₂₆ wasobserved by the end of 3 months.

In vivo Delivery of His-LEDGF₁₋₃₂₆ in Rats

In vivo delivery of His-LEDGF ₁₋₃₂₆ was evaluated following intravitrealadministration of Alexa Fluor 488 conjugated His-LEDGF₁₋₃₂₆ in NPinPMPin a rat model. No unlabeled LEDGF₁₋₃₂₆ was used in the NPinPMP. The rateyes were injected with Alexa-His-LEDGF₁₋₃₂₆ encapsulated NPinPMP (6.0μg of His-LEDGF₁₋₃₂₆) and as a control Alexa- His-LEDGF₁₋₃₂₆ =atequivalent concentration (1.5 μg labeled protein and 4.5 μg unlabeledprotein/5 μl) was injected. This ratio allowed us to start with asimilar fluorescence intensity for both groups to begin with. Ocularfluorescence due to the release of Alexa-His-LEDGF₁₋₃₂₆ was monitoredperiodically using Fluorotron Master™ (Ocumetrics, CA, USA) until thefluorescence reached the lower detection limit or baseline. Baselinefluorescence values of eyes were monitored before injecting theformulations. At each time point, three fluorometric scans were takenand mean value was used. Standard curve for Alexa- His-LEDGF₁₋₃₂₆ atdifferent concentrations was obtained using a cuvette and ocularflurophotometry with a rat lens adapter. The standard curve was used toconvert fluorescein equivalent concentrations provided byfluorophotometer to actual Alexa- His-LEDGF₁₋₃₂₆ concentration.

After intravitreal injection of Alexa-His-LEDGF₁₋₃₂₆ encapsulatingNPinPMP, and soluble Alexa- His-LEDGF_(1-326,) the concentrationsdistribution of His-LEDGF₁₋₃₂₆ along the eye optical axis was determinedindirectly by measuring the alexa fluorescence intensity distribution(equivalent of sodium fluoresciene concentration) curve along axialplanes, indicated as data points in an anterior to posterior direction.The fluorescence scans revealed sustained delivery ofAlexa-His-LEDGF₁₋₃₂₆ from NPinPMP compared to solution. Fluoresceinequivalent concentrations reported by Fluorotron Master were convertedto Alexa-His-LEDGF₁₋₃₂₆ concentrations. The Alexa-His-LEDGF₁₋₃₂₆concentration in the vitreous region from solution and NPinPMP group atdifferent time points was plotted. Only the concentrations of thelabeled bevacizumab are reported. Before intravitreal injection, thebaseline fluorescence readings of normal eyes were taken and thebaseline fluorescence concentration was found to be 2.03 μg/ml. Asshowing in FIGS. 26A-C, the Alexa-His-LEDGF₁₋₃₂₆ solution injected groupshowed Alexa- His-LEDGF₁₋₃₂₆ concentration of 2.02 μg/ml on day 1indicating rapid elimination from vitreous region. In NPinPMP injectedgroup the Alexa-His-LEDGF₁₋₃₂₆ the initial concentration in the vitreouswas found to be 18.23 μg/ml and the Alexa-His-LEDGF₁₋₃₂₆ concentrationabove the baseline was maintained until 35th day and reached normal baseline levels by end of 50 days. The observed data indicate the ability toachieve sustained in vivo release of Alexa- His-LEDGF₁₋₃₂₆ from anexemplary PinP composition.

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What is claimed is:
 1. A corticosteroid prodrug, comprising: i) acorticosteroid; and ii) a modification to the corticosteroid on eitherthe A, B, C, or D ring; wherein the modification increases thehydrophilicity of the corticosteroid.
 2. The prodrug according to claim1, wherein the modification is capable of having at least one negativelycharged terminal groups.
 3. The prodrug according to claim 2, whereinthe modification is chosen from sulphates, sulfphones, sulfoxides,sulphonic acids, citrates, phosphates, phosphines, phosphodiesters,phosphonic acids succinates, or salts thereof.
 4. The prodrug accordingto claim 1, wherein the modification is chosen from: i) —COCH₂COOH; ii)—COCH₂CH₂COOH; iii) —COCH₂CH₂CH₂COOH; iv) —COCH₂CH₂CH₂CH₂COOH; or v)—COCH₂CH(OH)COOH.
 5. The compound according to claim 1, wherein themodification has the formula:—PO(OH)_(2.)
 6. A therapeutic agent of the formula:

wherein R is a unit that increases the hydrophilicity of the compound.7. The compound according to claim 6, wherein R is chosen from sulphate,sulfphone, sulfoxide, sulphonic acis, citrate, phosphate, phosphine,phosphodiester, phosphonic acid succinate, or salts thereof.
 8. Thecompound according to claim 6, wherein R provides an ester of adi-carboxylic acid.
 9. The compound according to claim 8, wherein R ischosen from: i) —COCH₂COOH; ii) —COCH₂CH₂COOH; iii) —COCH₂CH₂CH₂COOH;iv) —COCH₂CH₂CH₂CH₂COOH; or v) —COCH₂CH(OH)COOH.
 10. The compoundaccording to claim 6, wherein the —COOH unit is further linked to ataurine.
 11. A composition for drug delivery, the compositioncomprising: a) an inner particle; and b) an outer particle; wherein thecomposition comprises a therapeutic agent of the formula:

wherein R is a unit that increases the hydrophobicity of the compound;wherein further the inner particle has a diameter of approximately 1 nmto approximately 999 nm, and wherein the outer particle has a diameterof approximately 1 to approximately 500 μm; and wherein the therapeuticagent is bound to the surface of the inner particle, bound to thesurface of the outer particle, encapsulated in the inner particle,encapsulated in the outer particle, present in the pores of the outerparticle or a combination thereof.
 12. The composition according toclaim 11, wherein the inner particle comprises polylactide (PLA),poly(glycolic acid) (PGA), co-polymer of lactic and glycolic acid(PLGA), cellulose derivatives, or chitosan.
 13. The compositionaccording to claim 12, wherein the inner particle comprises polylactide(PLA).
 14. The compositon according to claim 11, wherein the outerparticle comprises polyamides, polycarbonates, polyalkylene glycols,polyalkylene oxides, polyvinyl alcohols, polyvinyl ethers, polyvinylesters, polyvinylpyrrolidone, polyglycolides, and copolymers thereof,alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, polymers of acrylic and methacrylic esters,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,cellulose acetate cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose poly(methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate), poly(vinyl alcohols),poly(vinyl acetate), or polyvinylpryrrolidone.
 15. The compositionaccording to claim 11, wherein the outer particle compriseslactide-co-glycolide.
 16. The compound according to claim 11, wherein Ris chosen from sulphate, sulfphone, sulfoxide, sulphonic acis, citrate,phosphate, phosphine, phosphodiester, phosphonic acid succinate, orsalts thereof.
 17. The compound according to claim 11, wherein Rprovides an ester of a di-carboxylic acid.
 18. The compound according toclaim 17, wherein R is chosen from: i) —COCH₂COOH; ii) —COCH₂CH₂COOH;iii) —COCH₂CH₂CH₂COOH; iv) —COCH₂CH₂CH₂CH₂COOH; or v) —COCH₂CH(OH)COOH.19. A method for treating degenerative, vascular, infectious,angiogenic, and inflammatory diseases, comprising administering to asubject a prodrug according to claim
 1. 20. A method for treatingdegenerative, vascular, infectious, angiogenic, and inflammatorydiseases, comprising administering to a subject a composition accordingto claim 11.